Yeasts

J Cell Biol, 2005 Jan 17, 168(2), 257 - 69
Role of mitochondria in the pheromone- and amiodarone-induced programmed death of yeast; Pozniakovsky AI et al.; Although programmed cell death (PCD) is extensively studied in multicellular organisms, in recent years it has been shown that a unicellular organism, yeast Saccharomyces cerevisiae, also possesses death program(s) . In particular, we have found that a high doses of yeast pheromone is a natural stimulus inducing PCD . Here, we show that the death cascades triggered by pheromone and by a drug amiodarone are very similar . We focused on the role of mitochondria during the pheromone/amiodarone-induced PCD . For the first time, a functional chain of the mitochondria-related events required for a particular case of yeast PCD has been revealed: an enhancement of mitochondrial respiration and of its energy coupling, a strong increase of mitochondrial membrane potential, both events triggered by the rise of cytoplasmic {Ca(2+)}, a burst in generation of reactive oxygen species in center o of the respiratory chain complex III, mitochondrial thread-grain transition, and cytochrome c release from mitochondria . A novel mitochondrial protein required for thread-grain transition is identified.

J Cell Biol, 2005 Jan 17, 168(2), 209 - 19
Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase; Machin F et al.; Mitotic cell division involves the equal segregation of all chromosomes during anaphase . The presence of ribosomal DNA (rDNA) repeats on the right arm of chromosome XII makes it the longest in the budding yeast genome . Previously, we identified a stage during yeast anaphase when rDNA is stretched across the mother and daughter cells . Here, we show that resolution of sister rDNAs is achieved by unzipping of the locus from its centromere-proximal to centromere-distal regions . We then demonstrate that during this stretched stage sister rDNA arrays are neither compacted nor segregated despite being largely resolved from each other . Surprisingly, we find that rDNA segregation after this period no longer requires spindles but instead involves Cdc14-dependent rDNA axial compaction . These results demonstrate that chromosome resolution is not simply a consequence of compacting chromosome arms and that overall rDNA compaction is necessary to mediate the segregation of the long arm of chromosome XII.

J Biol Chem . 2005 Jan 18; {Epub ahead of print}
The role of the regulatory subunit of fission yeast calcineurin for in vivo activity and its relevance to FK506 sensitivity; Sio SO et al.; Calcineurin, a protein phosphatase required for Ca2+ signaling in many cell types, is a heterodimer composed of catalytic and regulatory subunits . The fission yeast genome encodes a single set of catalytic (Ppb1) and regulatory (Cnb1) subunits, providing an ideal model system to study these subunits' functions in vivo . Here, we cloned the cnb1+ gene, and showed that cnb1 knockout (Dcnb1) exhibits identical phenotypes with Dppb1, and that overexpression of Ppb1 failed to suppress the phenotypes of Dcnb1 . Interestingly, overexpression of C-terminal-deleted Ppb1 (Ppb1DC), the constitutively active form of Ppb1, also failed to suppress the phenotypes of Dcnb1 . FK506 caused MgCl2 sensitivity to the wild-type cells in an FKBP12-dependent manner . Co-overexpression of Ppb1 and Cnb1 suppressed the FK506-induced MgCl2 sensitivity, but the suppression was only partial, suggesting that an excess amount of Ppb1/Cnb1 complex cannot titrate out the FKBP12/FK506 complex . Although overexpression of Ppb1DC alone had little effect on cell growth, co-overexpression of Ppb1DC and Cnb1 caused distinct growth defect . FK506 suppressed the growth defect when Cnb1 was co-expressed using attenuated nmt1 promoter, but it failed to suppress the defect when Cnb1 was co-expressed using wild-type nmt1 promoter . Knockout of prz1+ gene, encoding a downstream target transcription factor of calcineurin, suppressed the growth defect irrespective of the promoter potency . These results suggest that Cnb1 is essential for the activation of calcineurin, and that the activated calcineurin is the pharmacological target of the FKBP12/FK506 complex in vivo.

J Biol Chem . 2005 Jan 18; {Epub ahead of print}
Search for apoptotic nucleases in yeast: Role of Tat-D nuclease in apoptotic DNA degradation; Qiu J et al.; DNA fragmentation/degradation is an important step for apoptosis . However, in unicellular organisms such as yeast, this process has rarely been investigated . In the current study, we revealed 8 apoptotic nuclease candidates in Saccharyomyces cerevisae, analogous to the Caenorhabditis elegans apoptotic nucleases . One of them is Tat-D . Sequence comparison indicates that Tat-D is conserved across kingdoms, implicating that it is evolutionarily and functionally indispensable . In order to better understand the biochemical and biological functions of Tat-D, we have overexpressed, purified and characterized the S . cerevisae Tat-D (scTat-D) . Our biochemical assays revealed that scTat-D is an endo-/exo-nuclease . It incises the double stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from 3' to 5' end by its exonuclease activity . The enzyme activities are metal dependent with Mg++ as an optimal metal ion and an optimal pH around 5 . We have also identified three amino acid residues H185, D325, and E327 important for its catalysis . In addition, our study demonstrated that knockout of TAT-D in S . cerevisae increases the TUNEL positive cells and cell survival in response to hydrogen hyperoxide treatment, whereas overexpression of Tat-D facilitates cell death . These results suggest a role of Tat-D in yeast apoptosis.

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 497 - 501
Geotrichum silvicola sp . nov., a novel asexual arthroconidial yeast species related to the genus Galactomyces; Pimenta RS et al.; Four strains of an asexual arthroconidial yeast species were isolated from Drosophila flies in two Atlantic rain forest sites in Brazil and two strains from oak tasar silkworm larvae (Antheraea proylei) in India . Analysis of the sequences of the D1/D2 large subunit rRNA gene showed that this yeast represented a novel species of the genus Geotrichum, described as Geotrichum silvicola sp . nov . The novel species was related to the ascogenous genus Galactomyces . The closest relatives of Geotrichum silvicola were Galactomyces sp . strain NRRL Y-6418 and Galactomyces geotrichum . The type culture of Geotrichum silvicola is UFMG-354-2(T) (=CBS 9194(T)=NRRL Y-27641(T)).

Gene, 2005 Jan 3, 344, 43 - 51 Epub 2004 Dec 10.
Drag&Drop cloning in yeast; Jansen G et al.; We have developed a set of vectors that have enhanced capabilities for efficiently constructing and expressing differentially tagged fusion proteins using Drag&Drop cloning in the yeast Saccharomyces cerevisiae . The pGREG vectors are based on the pRS series with an additional general kanR selection marker . In vivo homologous recombination is used to introduce genes of interest into galactose-inducible expression vectors (pGREGs), permitting the formation of amino-terminal fusions . The vectors all contain common regions for recombination that flank the stuffer fragment . Introduction of common recombination sequences at the end of PCR fragments will permit the cloning of genes without the need for specific restriction sites . In this process, the selectable stuffer HIS3 gene is replaced by successful gene integration, and a screen for loss of the selection marker identifies potential recombinants . Due to the modular structure of the vectors, genes introduced into one vector can be readily transferred by in vivo recombination to all other members of the vector system, thus permitting rapid and easy Drag&Drop construction of a series of tagged proteins . The pGREG series combines features for expression, tagging, integration, localization and library construction with the advantage of obtaining immediate results from sub-sequent experiments . This Drag&Drop system also allows efficient cloning and expression of heterologous genes in large-scale experiments.

FEBS J, 2005 Jan, 272(2), 524 - 37
Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening; Minic J et al.; The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding . Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae . The membrane expression of the receptor was shown by Western blotting and immunolocalization methods . Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter . Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis . The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system . The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands . The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional . Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.

Genome Res . 2005 Jan 14; {Epub ahead of print}
Genome-wide regulatory complexity in yeast promoters: Separation of functionally conserved and neutral sequence; Chin CS et al.; To gauge the complexity of gene regulation in yeast, it is essential to know how much promoter sequence is functional . Conservation across species can be a sensitive means of detecting functional sequences, provided that the significance of conservation can be accurately calibrated with the local neutral mutation rate . By analyzing yeast coding and promoter sequences, we find that neutral mutation rates in yeast are uniform genome-wide, in contrast to mammals, where neutral mutation rates vary along chromosomes . We develop an approach that uses this uniform rate to estimate the amount of promoter sequence under purifying selection . This amount is approximately 30%, corresponding to roughly 90 bp for a typical promoter . Furthermore, using a hidden Markov model, we are able to separate each promoter into distinct high and low conservation regions . Known regulatory motifs are strongly biased toward high conservation regions, while low conservation regions have mutation rates similar to that of the neutral background . Certain Gene Ontology groupings of genes (e.g., Carbohydrate Metabolism) have large amounts of high conservation sequence, suggesting complexity in their transcriptional regulation . Others (e.g., RNA Processing) have little high conservation sequence and are likely to be simply regulated . The separation of functionally conserved sequence from the neutral background allows us to estimate the complexity of cis-regulation on a genomic scale.

Trends Cell Biol, 2005 Jan, 15(1), 10 - 8
Split decisions: coordinating cytokinesis in yeast; Wolfe BA et al.; Cytokinesis in eukaryotes involves the regulated assembly and contraction of a ring comprising filamentous (F)-actin and myosin II . Assembly of the contractile ring occurs through the accumulation of cortical cues at the specified division plane, followed by recruitment of F-actin, myosin II and accessory proteins involved in generating the mature ring . Ring contraction is temporally regulated to occur only after chromosome segregation and, in yeast, it is controlled by a conserved signaling cascade that becomes active only after Cdk1-Cyclin-B inactivation . In this article (which is part of the Cytokinesis series), we discuss recent studies that have begun to clarify both the spatial and the temporal order of ring assembly and that have illuminated the signals that trigger ring contraction in yeast . These studies add to the growing knowledge of the processes that control eukaryotic cell division.

Cell, 2005 Jan 14, 120(1), 37 - 48
Mechanism of transcriptional silencing in yeast; Chen L et al.; Transcriptional silencing is a phenomenon in which the transcription of a gene by RNA polymerase II or III is repressed or not, dependent only on the gene's chromosomal location . Two prevailing models exist for silencing: (1) steric hindrance in silenced chromatin inhibits the binding of upstream activator proteins or polymerase or (2) silencing primarily blocks steps downstream of transcription preinitiation complex formation . Here, we test these models quantitatively for the case of SIR2-dependent silencing in budding yeast, using foreign and endogenous reporter proteins, at transgenic and endogenous loci . Our results contradict both models and show instead that transcriptional silencing at several URA3 transgenes, and at the naturally silenced endogenous HMRa and HMLalpha mating type genes, acts downstream of gene activator protein binding to strongly reduce the occupancy of TFIIB, RNA polymerase II, and TFIIE at the silenced promoters.

Cytometry A . 2005 Jan 13; {Epub ahead of print}
Detecting protein-protein interaction in live yeast by flow cytometry; Dye BT et al.; BACKGROUND: The yeast Saccharomyces cerevisiae is the most commonly used organism for studying protein- protein interactions . In this report we demonstrate the use of flow cytometry in observing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent fusion proteins (CFP and YFP, respectively) as a marker for protein interaction in live yeast cells . Probability binning is also employed to provide a statistical confirmation of our observations . METHODS: We coexpressed CFP and YFP fusions containing the N-terminal transmembrane domain (NTM) of Tom70p in yeast and analyzed FRET in live cells with a multilaser flow cytometer . The Tom70p NTM was previously shown to be sufficient for mitochondrial localization and protein-protein interaction (Millar and Shore, 1994, J Biol Chem 269:12229-12232) . RESULTS: FRET was observed only in cells that expressed CFP and YFP fusions that each contained the wild-type NTM . The introduction of mutations previously shown to disrupt NTM interaction eliminated FRET . Probability binning confirmed that differences between the FRET channels of experimental and control samples were statistically and physiologically significant . CONCLUSION: Flow cytometric analysis of FRET in yeast is a powerful technique for studying protein-protein interactions . The use of flow cytometry allows FRET data to be gathered from a large number of individual cells, thus providing important advantages unavailable to other techniques . Its application to yeast presents a new method to a popular system widely used in proteomic studies . (c) 2005 Wiley-Liss, Inc.

J Biol Chem . 2005 Jan 13; {Epub ahead of print}
Activation of the iron regulon by the yeast Aft1/Aft2 transcription factors depends on mitochondrial, but not cytosolic iron-sulfur protein biogenesis; Rutherford JC et al.; Two transcriptional activators, Aft1 and Aft2, regulate iron homeostasis in Saccharomyces cerevisiae . These factors induce the expression of iron regulon genes in iron-deficient yeast, but are inactivated in iron-replete cells . Iron inhibition of Aft1/Aft2 is abrogated in cells defective for Fe-S cluster biogenesis within the mitochondrial matrix (Chen et al . J . Biol . Chem . 279, 29513) . To determine whether iron sensing by Aft1/Aft2 requires the function of the mitochondrial Fe-S export and cytosolic Fe-S protein assembly systems, we evaluated the expression of the iron regulon in cells depleted of glutathione and in cells depleted of Atm1, Nar1, Cfd1 and Nbp35 . The iron regulon is induced in cells depleted of Atm1 with Aft1 being largely responsible for the induced gene expression . Aft2 is activated at a later time in Atm1 depleted cells . Likewise, the iron regulon is induced in cells depleted of glutathione . In contrast, repression of NAR1, CFD1 or NBP35 fails to induce the iron regulon despite strong inhibition of cytosolic/nuclear Fe-S protein assembly . Thus, iron sensing by Aft1/Aft2 is not linked to the maturation of cytosolic/ nuclear Fe-S proteins, but the mitochondrial inner membrane transporter Atm1 is important to transport the inhibitory iron signal . Although Aft1 and Aft2 sense a signal emanating from the Fe-S cluster biogenesis pathway, there is no indication that the proteins are inhibited by direct binding of an Fe-S cluster.

Biochem Biophys Res Commun, 2005 Feb 18, 327(3), 952 - 9
The yeast multidrug transporter Qdr3 (Ybr043c): localization and role as a determinant of resistance to quinidine, barban, cisplatin, and bleomycin; Tenreiro S et al.; Saccharomyces cerevisiae ORF YBR043c, predicted to code for a transporter of the major facilitator superfamily required for multiple drug resistance, encodes a plasma membrane protein that confers resistance to quinidine and barban, as observed before for its close homologues QDR1 and QDR2 . This ORF was, thus, named the QDR3 gene . The increased expression of QDR3, or QDR2, also leads to increased resistance to the anticancer agents cisplatin and bleomycin . However, no evidence for increased QDR3 expression in yeast cells exposed to all these inhibitory compounds was found . Transport assays support the concept that Qdr3 is involved, even if opportunistically, in the active export of quinidine out of yeast cell . A correlation was established between the efficiency of quinidine active export mediated by Qdr3p, Qdr2p or Qdr1p, and the efficacy of the expression of the encoding genes in alleviating the deleterious action of quinidine, as well as of the other compounds (QDR2>QDR3>>>QDR1).

Curr Biol, 2005 Jan 11, 15(1), 68 - 72
Yeast Nhp6A/B and Mammalian Hmgb1 Facilitate the Maintenance of Genome Stability; Giavara S et al.; Saccharomyces cerevisiae Nhp6A and Nhp6B are chromatin architectural factors that belong to the high-mobility group box (HMGB) superfamily and appear to be functionally related to mammalian Hmgb1 . They bind to the minor groove of double-stranded DNA in a non-sequence-specific manner and thereby influence chromatin structure . Previous work has implicated these proteins in a variety of nuclear processes, including chromatin remodeling, DNA replication, transcription, and recombination . Here, we show that Nhp6A/B loss leads to increased genomic instability, hypersensitivity to DNA-damaging agents, and shortened yeast cell life span that is associated with elevated levels of extrachromosomal rDNA circles . Furthermore, we show that hypersensitivity toward UV light does not appear to reflect a decreased capacity for DNA repair but instead correlates with higher levels of UV-induced thymine dimer adducts being formed in cells lacking Nhp6A/B . Likewise, we show that mouse fibroblasts lacking Hmgb1 display higher rates of damage after UV irradiation than wild-type controls and also exhibit pronounced chromosomal instability . Taken together, these data indicate that Nhp6A/B and Hmgb1 protect DNA from damaging agents and thus guard against the generation of genomic aberrations.

Biochem J . 2004 Dec 23; {Epub ahead of print}
The yeast cyclin-dependent kinase inhibitor Sic1 and the mammalian p27(Kip1) are functional homologues with a structurally conserved inhibitory domain; Barberis M et al.; In Saccharomyces cerevisiae, Sic1, an inhibitor of cyclin-dependent kinase, blocks the activity of Cdk1/Clb5,6 (S-Cdk1) kinase required for DNA replication . Deletion of Sic1 causes premature DNA replication from fewer origins, extension of the S phase and inefficient separation of sister chromatids during anaphase . Despite the well documented relevance of Sic1 inhibition of S-Cdk1 for cell cycle control and genome instability, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains obscure . In this report, we show that Sic1 is functionally and structurally related to mammalian cyclin-dependent kinase inhibitor (Cki) p27(Kip1) of the Kip/Cip family . A molecular model of the inhibitory domain of Sic1 bound to Cdk2/cyclin A complex suggested that the yeast inhibitor might productively interface with Cdk2/cyclin A mammalian complex . Consistently, Sic1 is able to bind to, and strongly inhibit the kinase activity of, Cdk2/cyclin A mammalian complex . Besides, the comparison of the different inhibitory patterns obtained using histone H1 or GST-pRb as substrates, the last one of which recognizes both the docking site and the catalytic site of Cdk2/cyclin A, offers interesting suggestions on the inhibitory mechanism of Sic1 . Finally, overexpression of the KIP1 gene in vivo in Saccharomyces cerevisiae rescues cell cycle-related phenotype of a sic1Delta strain, just as overexpression of the homologous SIC1 gene . Taken together, these findings strongly indicate that budding yeast Sic1 and mammalian p27(Kip1) are functional homologues with a structurally conserved inhibitory domain.

Mol Biol Cell . 2005 Jan 12; {Epub ahead of print}
Involvement of Sir2/4 in Silencing of DNA Breakage and Recombination on Mouse YACs during Yeast Meiosis; Klieger Y et al.; Monitoring Editor: Douglas Koshland Yeast artificial chromosomes (YACs) that contain human DNA backbone undergo DNA double strand breaks (DSBs) and recombination during yeast meiosis at rates similar to the yeast native chromosomes . Surprisingly, YACs containing DNA covering a recombination hot spot in the mouse Major Histocompatibility Complex class III region do not show meiotic DSBs and undergo meiotic recombination at reduced levels . Moreover, segregation of these YACs during meiosis is seriously compromised . In meiotic yeast cells carrying the mutations sir2 or sir4, but not sir3, these YACs show DSBs, suggesting that a unique chromatin structure of the YACs, involving Sir2 and Sir4, protects the YACs from the meiotic recombination machinery . We speculate that the paucity of DSBs and recombination events on these YACs during yeast meiosis may reflect the refractory nature of the corresponding region in the mouse genome.

Mol Biol Cell . 2005 Jan 12; {Epub ahead of print}
Golgi-to-Late Endosome Trafficking of the Yeast Pheromone Processing Enzyme Ste13p Is Regulated by a Phosphorylation Site in its Cytosolic Domain; Johnston HD et al.; Monitoring Editor: Benjamin Glick This study addressed whether phosphorylation regulates trafficking of yeast membrane proteins that cycle between the trans-Golgi network (TGN) and endosomal system . The TGN membrane proteins A-alkaline phosphatase, a model protein containing the Ste13p cytosolic domain fused to alkaline phosphatase (ALP), and Kex2p were found to be phosphorylated in vivo . Mutation of the S13 residue on the cytosolic domain of A-alkaline phosphatase to Ala was found to block trafficking to the prevacuolar compartment (PVC) while a S13D mutation generated to mimic phosphorylation accelerated trafficking into the PVC . The S13 residue was shown by mass spectrometry to be phosphorylated . The rate of ER-to-Golgi transport of newly synthesized A(S13A)-alkaline phosphatase was indistinguishable from wild-type indicating that the lack of transport of A(S13A)-alkaline phosphatase to the PVC was instead due to differences in Golgi/endosomal trafficking . The A(S13A)-alkaline phosphatase protein exhibited a TGN-like localization similar to that of wild-type A-alkaline phosphatase . Similarly, the S13A mutation in endogenous Ste13p did not reduce the extent of or longevity of its localization to the TGN as shown by alpha-factor processing assays . These results indicate that S13 phosphorylation is required for TGN-to-PVC trafficking of A-alkaline phosphatase and imply that phosphorylation of S13 may regulate recognition of A-alkaline phosphatase by vesicular trafficking machinery.

Mol Biol Cell . 2005 Jan 12; {Epub ahead of print}
The Roles of Fission Yeast Ase1 in Mitotic Cell Division, Meiotic Nuclear Oscillation and Cytokinesis Checkpoint Signaling; Yamashita A et al.; Monitoring Editor: Tim Stearns The Ase1/Prc1 proteins constitute a conserved microtubule associated protein family that is implicated in central spindle formation and cytokinesis . Here we characterize a role for fission yeast Ase1 . Ase1 localizes to microtubule overlapping zones, and displays dynamic alterations of localization during the cell cycle . In particular, its spindle localization during metaphase is reduced substantially, followed by robust appearance at the spindle midzone in anaphase . ase1 deletions are viable but defective in nuclear and septum positioning and completion of cytokinesis, which leads to diploidization and chromosome loss . Time-lapse imaging shows that elongating spindles collapse abruptly in the middle of anaphase B . Either absence or overproduction of Ase1 results in profound defects on microtubule bundling in an opposed manner, indicating that Ase1 is a dose-dependent microtubule bundling factor . In contrast microtubule nucleating activities are not noticeably compromised in ase1 mutants . During meiosis astral microtubules are not bundled and oscillatory nuclear movement is impaired significantly . The Aurora kinase does not correctly localize to central spindles in the absence of Ase1 . Finally Ase1 acts as a regulatory component in the cytokinesis checkpoint that operates to inhibit nuclear division when the cytokinesis apparatus is perturbed . Ase1, therefore, couples anaphase completion with cytokinesis upon cell division.

Proc Natl Acad Sci U S A, 2005 Jan 18, 102(3), 707 - 12 Epub 2005 Jan 12.
Rapid evolution of expression and regulatory divergences after yeast gene duplication; Gu X et al.; Although gene duplication is widely believed to be the major source of genetic novelty, how the expression or regulatory network of duplicate genes evolves remains poorly understood . In this article, we propose an additive expression distance between duplicate genes, so that the evolutionary rate of expression divergence after gene duplication can be estimated through phylogenomic analysis . We have analyzed yeast genome sequences, microarrays, and transcriptional regulatory networks, showing a >10-fold increase in the initial rate for both expression and regulatory network evolution after gene duplication but only an approximately 20% rate increase in the early stage for protein sequences . Based on the estimated age distribution of yeast duplicate genes, we roughly estimate that the initial rate of expression divergence shortly after gene duplication is 2.9 x 10(-9) per year, whereas the baseline rate for very ancient gene duplication is 0.14 x 10(-9) per year . Relative expression rate tests suggest that the expression of duplicate genes tends to evolve asymmetrically, that is, the expression of one copy evolves rapidly, whereas the other one largely maintains the ancestral expression profile . Our study highlights the crucial role of early rapid evolution after gene/genome duplication for continuously increasing the complexity of the yeast regulatory network.

Yeast . 2005 Jan 11;22(2):99-110 {Epub ahead of print}
A new mutation in the yeast aspartate kinase induces threonine accumulation in a temperature-regulated way; Velasco I et al.; In Saccharomyces cerevisiae, aspartate kinase (the HOM3 product) regulates the metabolic flux through the threonine biosynthetic pathway through feedback inhibition by the end product . In order to obtain a strain able to produce threonine in a controlled way, we have isolated a mutant allele (HOM3-ts31d) that gives rise to a deregulated aspartate kinase . This allele has been isolated as an extragenic suppressor of ilv1, which confers an Ilv(+) phenotype at 37 degrees C but not at 22 degrees C . We have stated that at high temperature the mutant aspartate kinase is slightly more deregulated and shows a higher specific activity, inducing threonine accumulation . The HOM3-ts31d allele carries a mutation that leads to a Ser399 --> Phe substitution in the postulated regulatory region of the enzyme . We have detected other changes in the nucleotide sequence but they are also present in the parental strain, reflecting the genetic differences between different wild-type strains . A sequence comparison among all the reported mutant aspartate kinases suggests that not all residues involved in regulation of the activity are clustered in the so-called regulatory domain, as is the case of that mutated in AK-R7, another deregulated aspartate kinase obtained with the same strategy of ilv1 suppression . Copyright (c) 2005 John Wiley & Sons, Ltd.

J Biol Chem . 2005 Jan 11; {Epub ahead of print}
Phosphate carrier has an ability to be sorted to either the TIM22 pathway or TIM23 pathway for its import into yeast mitochondria; Yamano K et al.; Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria via the TOM40 complex, and follow several distinct sorting pathways to reach their destination submitochondrial compartments . Phosphate carrier (PiC) is an inner membrane protein with 6 transmembrane segments (TM1-TM6) and requires, after translocation across the outer membrane, the Tim9-Tim10 complex and the TIM22 complex to be inserted into the inner membrane . Here we analyzed in vitro import of fusion proteins between various PiC segments and mouse dihydrofolate reductase . The fusion protein without TM1-TM2 was translocated across the outer membrane, but was not inserted into the inner membrane . The fusion proteins without TM1-TM4 were not inserted into the inner membrane, but instead translocated across the inner membrane . Functional defects of Tim50 of the TIM23 complex either by depletion of the protein or addition of anti-Tim50 antibodies blocked translocation of the fusion proteins without TM1-TM4 across the inner membrane, suggesting that lack of TM1-TM4 led to switch of its sorting pathway from the TIM22 pathway to the TIM23 pathway . PiC thus appears to have a latent signal for sorting to the TIM23 pathway, which is exposed by reduced interactions with the Tim9-Tim10 complex and maintenance of the import competence.

FEBS Lett, 2005 Jan 17, 579(2), 559 - 66
Yeast two-hybrid screens imply that GGNBP1, GGNBP2 and OAZ3 are potential interaction partners of testicular germ cell-specific protein GGN1; Zhang J et al.; Gametogenetin (Ggn) is a testicular germ cell-specific gene specifically expressed from late pachytene spermatocytes through round spermatids . The function of gametogenetin protein 1 (GGN1) remains unknown . Here, we used the yeast two-hybrid approach to look for more GGN1 interacting proteins . We found that gametogenetin binding protein 1 (GGNBP1), gametogenetin binding protein 2 (GGNBP2) and ornithine decarboxylase antizyme 3 (OAZ3) were potential GGN1 interaction partners . We determined the regions mediating the interactions and further showed the interactions between the proteins in mammalian cells by colocalization and coimmunoprecipitation experiments . Our work suggested that GGN1, GGNBP1, GGNBP2 and OAZ3 could be involved in a common process associated with spermatogenesis.

FEBS Lett, 2005 Jan 17, 579(2), 512 - 6
A role for the non-phosphorylated form of yeast Snf1: tolerance to toxic cations and activation of potassium transport; Portillo F et al.; The Snf1/AMP-activated protein kinases play a key role in stress responses of eukaryotic cells . In the yeast Saccharomyces cerevisiae Snf1 is regulated by glucose depletion, which triggers its phosphorylation at Thr210 and concomitant increase in activity . Activated yeast Snf1 is required for the metabolic changes allowing starvation tolerance and utilization of alternative carbon sources . We now report a function for the non-activated form of Snf1: the regulation of the Trk high-affinity potassium transporter, encoded by the TRK1 and TRK2 genes . A snf1Delta strain is hypersensitive in high-glucose medium to different toxic cations, suggesting a hyperpolarization of the plasma membrane driving increased cation uptake . This phenotype is suppressed by the TRK1 and HAL5 genes in high-copy number consistent with a defect in K(+) uptake mediated by the Trk system . Accordingly, Rb(+) uptake and intracellular K(+) measurements indicate that snf1Delta is unable to fully activate K(+) import . Genetic analysis suggests that the weak kinase activity of the non-phosphorylated form of Snf1 activates Trk in glucose-metabolizing yeast cells . The effect of Snf1 on Trk is probably indirect and could be mediated by the Sip4 transcriptional activator.

FEBS Lett, 2005 Jan 17, 579(2), 507 - 11
Identification of a novel member of yeast mitochondrial Hsp70-associated motor and chaperone proteins that facilitates protein translocation across the inner membrane; Yamamoto H et al.; Here, we report the identification of yeast 15-kD Tim15/Zim17, a new member of mitochondrial Hsp70 (mtHsp70)-associated motor and chaperone (MMC) proteins . The 15-kD MMC protein is a peripheral inner membrane protein with a zinc-finger motif . Depletion of the 15-kD protein led to impaired import of presequence-containing proteins into the matrix in vivo and in vitro . Overexpression of the 15-kD protein rescued the functional defects of mtHsp70 in ssc1-3 cells, and a fusion protein containing the 15-kD protein physically interacts with purified mtHsp70 . Tim15/Zim17 therefore cooperates with mtHsp70 to facilitate import of presequence-containing proteins into the matrix.

Ai Zheng, 2005 Jan, 24(1), 40 - 6
{Screening ubiquitin/ribosomal protein s27a by yeast two-hybridization system.}; Huang GH et al.; BACKGROUND & OBJECTIVE: Protein kinase CK2, a kind of ubiquitous eukaryotic messenger-independent protein serine/threonine kinase, plays a vital role in cell differentiation and proliferation, signal transduction and procession . Activity of CK2 in hematopoietic cells is 2-8 folds higher than that in relevant normal tissues, moreover changes of CK2 activity are correlated to tumor growth . In order to investigate the mechanism of its effect on hematopoietic cells, we used yeast two-hybridization screening the proteins interacting with protein kinase CK2alpha' subunit from HL-60 cells cDNA library . METHODS: The target CK2alpha' cDNA was obtained by amplifying recombinant plasmid pTHCK2A', containing human protein kinase CK2alpha' subunit cDNA, through polymerase chain reaction (PCR) . Pst I/Nde I-digested PCR products were directionally cloned into DNA-BD vector pGBKT7, which had also been digested by Pst I/Nde I . The recombinant plasmid was named yeast two-hybridization BD bait plasmid, and confirmed by DNA sequencing, and auto-activated experiments . Total RNA of HL-60 cells was extracted, CLONTECH switching mechanism at 5' end of RNA transcirpt method was used to construct a cDNA library in yeast cells . Library plasmid was named AD plasmid . BD plasmid and AD plasmid were co-transformed into competent yeast AH109 . Yeast two-hybridization was used to screen positive clones . RESULTS: Six proteins, interacting with human protein kinase CK2alpha' subunit, were screened . DNA sequencing and homology comparison showed that one of the proteins was highly homologous with ubiquitin/ribosomal protein S27a (99.8%) . CONCLUSION: Using yeast two-hybridization system could screen out ubiquitin/ribosomal protein S27a, which may interact with human protein kinase CK2alpha' subunit, from HL-60 cells.

Oncogene, 2005 Jan 10, 24(2), 217 - 29
Yeast polo-like kinases: functionally conserved multitask mitotic regulators; Lee KS et al.; The polo-like kinases (Plks) are a conserved subfamily of Ser/Thr protein kinases that play pivotal roles in regulating various cellular and biochemical events at multiple stages of M phase . Genetic and biochemical data revealed that both the budding yeast and the fission yeast polo kinase homologs (Cdc5 and Plo1, respectively) bear remarkable functional similarities with those in metazoan organisms, suggesting that the role of Plks is largely conserved throughout evolution . Thus, studies on Plks in genetically amenable lower eucaryotic organisms may yield valuable insights into the function of Plks in higher eucaryotic organisms . In this review, common properties and distinct functions of Cdc5 and Plo1 will be discussed and compared to properties and functions of Plks in higher eucaryotic organisms.

Appl Environ Microbiol, 2005 Jan, 71(1), 312 - 9
Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis; Hashimoto S et al.; Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes . We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis . We isolated His(-), Met(-), Lys(-), Trp(-), Leu(-), Arg(-), and Ura(-) auxotrophic mutants of five sake strains, Kyokai no . 7, no . 9, no . 10, no . 701, and no . 901, by screening only 1,700 to 3,400 colonies from each treated strain . Wild-type alleles were cloned and used as markers for transformation . With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no . 7 . The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor . We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.

J Biol Chem . 2005 Jan 6; {Epub ahead of print}
The binding mechanism of the yeast F1-ATPase inhibitory peptide . Role of catalytic intermediates and enzyme turnover; Corvest V et al.; The mechanism of inhibition of yeast mitochondrial F1-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites . Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Tyr345Trp) . Fluorescence quenching of a beta-Trp345 indicates that the binding of MgADP to F1 can be described as 3 binding sites with dissociation constants of Kd1=10+/-2 nM, Kd2=0.22+/-0.03 microM, Kd3=16.3+/-0.2 microM . In addition, the ATPase activity of the beta-Trp345 enzyme followed simple Michaelis-Menten kinetics with a corresponding Km of 55 microM . Values for the Kd for MgATP were estimated and indicate that the Km (55 microM) for ATP hydrolysis corresponds to filling the third catalytic site on F1 . IF1 binds very slowly to F1-ATPase depleted of nucleotides and under unisite conditions . The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 microM, but decreased thereafter . The rate of inhibition was half-maximal at 5 microM MgATP, which is 10 fold lower than the Km for ATPase . The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis . A model is proposed that suggests that IF1 binds rapidly, but loosely to F1 with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.

Lipids, 2004 Aug, 39(8), 747 - 52
A mutation in sphingolipid synthesis suppresses defects in yeast ergosterol metabolism; Valachovic M et al.; A mutation in an otherwise nonessential ERG2 gene is synthetically lethal when combined with mutations in two transcription factors encoded by the UPC2 and ECM22 genes . Employing UV mutagenesis, we isolated a suppressor of the triple mutant erg2delta upc2delta ecm22delta . The morpholine-resistant phenotype of the suppressor was used to identify the suppressor as a mutation in the ELO3 gene . In an expression study on tridemorph-containing medium, using the inducible GAL1 promoter fused to the ELO3 open reading frame, we demonstrated that suppression occurred only when ELO3 was not expressed . ELO3 encodes an enzyme involved in sphingolipid synthesis required for long-chain FA synthesis . Surprisingly, a deletion of ELO2, also required for the synthesis of sphingolipid-containing long-chain FA, did not suppress the erg2delta upc2delta ecm22delta triple mutant . The sterol composition of the upc2delta ecm22delta double mutant reflected regulation of the latter part of the ergosterol synthesis by the Upc2p and Ecm22p transcription factors . This study demonstrates a synergistic relationship between two lipid species, sterols and sphingolipids.

J Biol Chem . 2005 Jan 6; {Epub ahead of print}
Synthase III-dependent chitin is bound to different acceptors depending on location on the cell wall of budding yeast; Cabib E et al.; In yeast, chitin is laid down at three locations: a ring at the mother-bud neck, the primary septum and, after cytokinesis, the cell wall of the daughter cell . Some of the chitin is free, the remainder attached to beta(1-3)glucan or beta(1-6)glucan . We recently reported that the chitin ring contributes to the prevention of growth at the mother-bud neck and hypothesized that this inhibition is achieved by a preferential binding of chitin to beta(1-3)glucan at that site . Here, we devised a novel strategy for the analysis of chitin cross-links in 14C-glucosamine-labeled cell walls, involving solubilization in water of alkali-treated walls by carboxymethylation . Intact cell walls or their digestion products with beta(1-3)glucanase or beta(1-6)glucanase were carboxymethylated and fractionated on size columns, and the percentage of chitin bound to different polysaccharides was calculated . Chitin dispersed in the wall was labeled in maturing unbudded cells and that of the ring in early budding cells . The former was mostly attached to beta(1-6)glucan and the latter to beta(1-3)glucan . This confirmed our hypothesis and indicated that the cell has mechanisms to attach chitin, a water-insoluble substance, synthesized here through chitin synthase III, to different acceptors, depending on location . In contrast, most of the chitin synthase II-dependent chitin of the primary septum was free, with the remainder linked to beta(1-3)glucan.

J Biol Chem . 2005 Jan 6; {Epub ahead of print}
Extended DNA binding site in Pot1 broadens sequence specificity to allow recognition of heterogeneous fission yeast telomeres; Trujillo KM et al.; The Pot1 (protection of telomeres) protein binds to single-stranded telomeric DNA and is essential for the protection of chromosome ends from degradation and end-to-end fusions . The Pot1 amino-terminal DNA-binding domain, Pot1N, adopts an oligonucleotide/oligosaccharide-binding fold and binds GGTTAC motifs cooperatively and with exceptionally high sequence specificity . We have now examined DNA-binding to naturally occurring telomeric substrates based on the analysis of 100 cloned chromosome ends and in the context of the full-length Pot1 protein . Here, we describe several important differences between Pot1 and Pot1N with apparent consequences for chromosome end protection . Specifically, full length Pot1-DNA complexes are more stable and the minimal binding site for a Pot1 monomer is extended into two adjacent telomeric repeats . We provide evidence that Pot1 contains a second DNA-binding motif that recognizes DNA with reduced sequence specificity compared to the domain present in Pot1N . The two DNA-binding motifs cooperate, whereby the amino-terminal OB-fold determines the registry of binding and the internal DNA-binding motif stabilizes the complex and expands the protected region towards the 3' end . Consistent with a role in chromosome end capping, Pot1 prevents access of telomerase to the 3' end and protects against exonucleolytic degradation.

Mol Biol Cell . 2005 Jan 5; {Epub ahead of print}
The Yeast Endosomal Na+(K+)/H+ Exchanger Nhx1 Regulates Cellular pH to Control Vesicle Trafficking; Brett CL et al.; Monitoring Editor: Sandra Schmid The relationship between endosomal pH and function is well documented in viral entry, endosomal maturation, receptor recycling, and vesicle targeting within the endocytic pathway . However, specific molecular mechanisms that either sense or regulate luminal pH to mediate these processes have not been identified . Herein we describe the use of novel, compartment-specific pH indicators to demonstrate that yeast Nhx1, an endosomal member of the ubiquitous NHE family of Na(+)/H(+) exchangers, regulates luminal and cytoplasmic pH to control vesicle trafficking out of the endosome . Loss of Nhx1 confers growth sensitivity to low pH stress, and concomitant acidification and trafficking defects, which can be alleviated by weak bases . Conversely, weak acids cause wild-type yeast to present nhx1Delta trafficking phenotypes . Finally, we report that Nhx1 transports K(+) in addition to Na(+), suggesting that a single mechanism may responsible for both pH and K(+)-dependent endosomal processes . This presents the newly defined family of eukaryotic endosomal NHE as novel targets for pharmacological inhibition to alleviate pathological states associated with organellar alkalinization.

J Am Anim Hosp Assoc, 2005 Jan-Feb, 41(1), 12 - 21
Efficacy of boric-complexed zinc and acetic-complexed zinc otic preparations for canine yeast otitis externa; Mendelsohn CL et al.; The purpose of this 2-week, double-blinded, controlled clinical trial was to evaluate the efficacy of topical amino acid-complexed zinc gluconate formulated with boric acid (ZGB) or acetic acid (ZGA) versus a topical placebo in the treatment of yeast otitis externa in dogs . Included in the study were dogs with otitis externa and a cytopathological finding of yeast organisms in the affected ear . Ears were treated with the placebo, ZGA, or ZGB medications . Yeast counts as well as clinical appearance of the ears were monitored . Results revealed that ZGB significantly reduced the number of yeast organisms in cases of otitis externa.

Cell Stress Chaperones, 2004 Winter, 9(4), 359 - 68
Investigating the protein-protein interactions of the yeast Hsp90 chaperone system by two-hybrid analysis: potential uses and limitations of this approach; Millson SH et al.; The Hsp90 chaperone cycle involves sequential assembly of different Hsp90-containing multiprotein complexes, the accessory proteins ("cochaperones") that are associated with these complexes being exchanged as the cycle proceeds from its early to its late stages . To gain insight as to whether the 2-hybrid system could be used to probe the interactions of this Hsp90 system, yeast transformants were constructed that express the Gal4p deoxyribonucleic acid-binding domain (BD) fused to the 2 Hsp90 isoforms and the various Hsp90 system cochaperones of yeast . These "bait" fusions were then introduced by mating into other transformants expressing nearly all the 6000 proteins of yeast expressed as fusions to the Gal4p activation domain (AD) . High throughput 2-hybrid screening revealed the ability of Hsp90 and Hsp90 system cochaperones to engage in stable interactions in vivo, both with each other and with the various other proteins of the yeast proteome . Consistent with the transience of most chaperone associations, interactions to Hsp90 itself were invariably weak and generally influenced by stress . Mutations within a Hsp90-BD bait fusion and an AD-Cdc37 "prey" fusion were used to provide in vivo confirmation of the in vitro data that shows that Cdc37p is interacting with the "relaxed" conformation of Hsp90 and also to provide indications that Cdc37p needs to be phosphorylated at its N-terminus for any appreciable interaction with Hsp90 . A number of potentially novel cochaperone interactions were also identified, providing a framework for these to be analyzed further using other techniques.

Am J Pathol, 2005 Jan, 166(1), 265 - 74
A human yeast artificial chromosome containing the multiple endocrine neoplasia type 2B Ret mutation does not induce medullary thyroid carcinoma but does support the growth of kidneys and partially rescues enteric nervous system development in Ret-deficient mice; Skinner MA et al.; We generated a line of transgenic mice using a yeast artificial chromosome containing the Ret mutation responsible for the multiple endocrine neoplasia type 2B syndrome (MEN 2B) . The resulting animals did not develop any of the expected neoplasms associated with MEN 2B . Transgenic animals were then bred with animals lacking murine Ret (Ret(M)) to further evaluate the function of human mutated Ret (Ret(H)(2B)) in the murine context . Whereas mice lacking Ret(M) exhibit intestinal aganglionosis and the absence of kidneys with other genitourinary anomalies, expression of the Ret(H)(2B) transgene in Ret(M)-deficient mice allowed significant renal development with a partial rescue of the enteric nervous system . These Ret(H)(2B)-positive/Ret(M)-deficient mice exhibit normal Ret expression and survive longer than Ret(M)-deficient mice, but still die at 3 to 5 days of age with evidence of enterocolitis . We conclude that the normal expression of a human Ret proto-oncogene with the MEN 2B mutation does not cause any features of MEN 2B in mice . Although the gene is normally expressed in the appropriate target tissues, there is incomplete phenotypic rescue in mice lacking murine Ret . These results suggest important interspecies differences between humans and mice in the function of the Ret oncogene.

J Cell Biol, 2005 Jan 3, 168(1), 103 - 15
Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast; Dimmer KS et al.; The MDM31 and MDM32 genes are required for normal distribution and morphology of mitochondria in the yeast Saccharomyces cerevisiae . They encode two related proteins located in distinct protein complexes in the mitochondrial inner membrane . Cells lacking Mdm31 and Mdm32 harbor giant spherical mitochondria with highly aberrant internal structure . Mitochondrial DNA (mtDNA) is instable in the mutants, mtDNA nucleoids are disorganized, and their association with Mmm1-containing complexes in the outer membrane is abolished . Mutant mitochondria are largely immotile, resulting in a mitochondrial inheritance defect . Deletion of either one of the MDM31 and MDM32 genes is synthetically lethal with deletion of either one of the MMM1, MMM2, MDM10, and MDM12 genes, which encode outer membrane proteins involved in mitochondrial morphogenesis and mtDNA inheritance . We propose that Mdm31 and Mdm32 cooperate with Mmm1, Mmm2, Mdm10, and Mdm12 in maintenance of mitochondrial morphology and mtDNA.

J Am Chem Soc, 2005 Jan 12, 127(1), 92 - 9
EPR and optical spectroscopic studies of Met80X mutants of yeast ferricytochrome c . Models for intermediates in the alkaline transition; Silkstone GG et al.; The ferric forms of Met80X mutants of yeast iso-1-cytochrome c (X = Ala, Ser, Asp, and Glu) display EPR and optical spectra that are strongly pH dependent . At low pH values (pH approximately 5) the sixth coordination sites are filled by H(2)O that, on elevating the pH, is replaced by OH(-) in the cases of Met80Ala and -Ser (pK approximately 5.6 and 5.9, respectively) and by a lysine amino group in the cases of Met80Asp and -Glu (pK approximately 9.3 and 11.6, respectively) . The ligand sets and the pK values of the transitions are rationalized in terms of the structure of the heme pocket, and a possible mechanism of the "trigger" in the alkaline transition of the native protein is suggested.

Genes Dev, 2005 Jan 1, 19(1), 90 - 103
Yeast poly(A)-binding protein, Pab1, and PAN, a poly(A) nuclease complex recruited by Pab1, connect mRNA biogenesis to export; Dunn EF et al.; In eukaryotic cells, pre-mRNAs undergo extensive processing in the nucleus prior to export . Processing is subject to a quality-control mechanism that retains improperly processed transcripts at or near sites of transcription . A poly(A) tail added by the normal 3'-processing machinery is necessary but not sufficient for export . Retention depends on the exosome . In this study, we identify the poly(A)-binding protein, Pab1, and the poly(A) nuclease, PAN, as important factors that couple 3' processing to export . Pab1 contains a nonessential leucine-rich nuclear export signal and shuttles between the nucleus and the cytoplasm . It can exit the nucleus either as cargo of exportin 1 or bound to mRNA . Pab1 is essential but several bypass suppressors have been identified . Deletion of PAB1 from these bypass suppressor strains results in exosome-dependent retention at sites of transcription . Retention is also seen in cells lacking PAN, which Pab1 is thought to recruit and which may be responsible for the final step of mRNA biogenesis, trimming of the poly(A) tail to the length found on newly exported mRNAs . The studies presented here suggest that proper loading of Pab1 onto mRNAs and final trimming of the tail allows release from transcription sites and couples pre-mRNA processing to export.

Mol Cell, 2005 Jan 7, 17(1), 49 - 59
Inactivation of the Cdc25 Phosphatase by the Stress-Activated Srk1 Kinase in Fission Yeast; Lopez-Aviles S et al.; The mechanisms by which environmental stress regulates cell cycle progression are poorly understood . In fission yeast, we show that Srk1 kinase, which associates with the stress-activated p38/Sty1 MAP kinase, regulates the onset of mitosis by inhibiting the Cdc25 phosphatase . Srk1 is periodically active in G2, and its overexpression causes cell cycle arrest in late G2 phase, whereas cells lacking srk1 enter mitosis prematurely . We find that Srk1 interacts with and phosphorylates Cdc25 at the same sites phosphorylated by the Chk1 and Cds1 (Chk2) kinases and that this phosphorylation is necessary for Srk1 to delay mitotic entry . Phosphorylation by Srk1 causes Cdc25 to bind to Rad24, a 14-3-3 protein family member, and accumulation of Cdc25 in the cytoplasm . However, Srk1 does not regulate Cdc25 in response to replication arrest or DNA damage but, rather, during a normal cell cycle and in response to nongenotoxic environmental stress.

J Struct Biol, 2005 Jan, 149(1), 111 - 5
Gene silencing pathway RNA-dependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein; Laurila MR et al.; The RNA-dependent RNA polymerase, QDE-1, is a component of the RNA silencing pathway in Neurospora crassa . The enzymatically active carboxy-terminal fragment QDE-1 DeltaN has been expressed in Saccharomyces cerevisiae in the presence and absence of selenomethionine (SeMet) . The level of SeMet incorporation was estimated by mass spectrometry to be approximately 98% . Both native and SeMet proteins were crystallized in space group P2(1) with unit cell parameters a=101.2, b=122.5, c=114.4A, beta=108.9 degrees , and 2 molecules per asymmetric unit . The native and SeMet crystals diffract to 2.3 and 3.2A, respectively, the latter are suitable for MAD structure determination.

Biochem Biophys Res Commun, 2005 Feb 4, 327(1), 143 - 9
Identification of an evolutionary conserved SURF-6 domain in a family of nucleolar proteins extending from human to yeast; Polzikov M et al.; The mammalian SURF-6 protein is localized in the nucleolus, yet its function remains elusive in the recently characterized nucleolar proteome . We discovered by searching the Protein families database that a unique evolutionary conserved SURF-6 domain is present in the carboxy-terminal of a novel family of eukaryotic proteins extending from human to yeast . By using the enhanced green fluorescent protein as a fusion protein marker in mammalian cells, we show that proteins from distantly related taxonomic groups containing the SURF-6 domain are localized in the nucleolus . Deletion sequence analysis shows that multiple regions of the SURF-6 protein are capable of nucleolar targeting independently of the evolutionary conserved domain . We identified that the Saccharomyces cerevisiae member of the SURF-6 family, named rrp14 or ykl082c, has been categorized in yeast databases to interact with proteins involved in ribosomal biogenesis and cell polarity . These results classify SURF-6 as a new family of nucleolar proteins in the eukaryotic kingdom and point out that SURF-6 has a distinct domain within the known nucleolar proteome that may mediate complex protein-protein interactions for analogous processes between yeast and mammalian cells.

Biochem Biophys Res Commun, 2005 Feb 11, 327(2), 597 - 603
Random mutagenesis of the B'A' core domain of yeast DNA topoisomerase II and large-scale screens of mutants resistant to the anticancer drug etoposide; Jiang X; Mutagenic PCR method was applied to introduce point mutations to the B'A' core domain of yeast DNA topoisomerase II . Screens for mutants resistant to the anticancer drug etoposide were carried out in a yeast ts system in the presence of high concentrations of the drug or in a drug-hypersensitive genetic background . 129 mutants were obtained from a total of 47,000 transformants . Nucleotide sequencing of 40 selected mutants showed that a large number of the mutations map to regions encoding the linker that joins the ATPase domain to the B' module and the B'A' linker . Significant reduction in catalytic activity was evident for a large fraction of mutant enzymes and all mutants were also resistant to amsacrine, another topoisomerase II drug with a different chemical structure, suggesting that few of the mutations reflect simple changes of specific amino acid side chains that are directly involved in enzyme-drug interactions.

Biochemistry, 2005 Jan 11, 44(1), 377 - 86
Kinetic Analysis of YPD1-Dependent Phosphotransfer Reactions in the Yeast Osmoregulatory Phosphorelay System; Janiak-Spens F et al.; In Saccharomyces cerevisiae, the histidine-containing phosphotransfer (HPt) protein YPD1 transfers phosphoryl groups between the three different response regulator domains of SLN1, SSK1, and SKN7 (designated R1, R2, and R3, respectively) . Together these proteins form a branched histidine-aspartic acid phosphorelay system through which cells can respond to hyperosmotic and other environmental stresses . The in vivo order of phosphotransfer reactions is believed to proceed from SLN1-R1 to YPD1 and then subsequently to SSK1-R2 or SKN7-R3 . The individual phosphoryl transfer reactions between YPD1 and the response regulator domains have been examined kinetically . A maximum forward rate constant of 29 s(-)(1) was determined for the reaction between SLN1-R1 approximately P and YPD1 with a K(d) of 1.4 muM for the SLN1-R1 approximately P.YPD1 complex . In the subsequent reactions, phosphotransfer from YPD1 to SSK1-R2 is very rapid (160 s(-)(1)) and is strongly favored over phosphotransfer to SKN7-R3 . Phosphotransfer reactions between YPD1 and SLN1-R1 or SKN7-R3 were reversible . In contrast, no reverse transfer from SSK1-R2 approximately P to YPD1 was observed . These findings are consistent with the notion that SSK1 is constitutively phosphorylated under normal osmotic conditions . In addition, we have examined the roles of several conserved amino acid residues surrounding the phosphorylatable histidine (H64) of YPD1 using phosphoryl transfer reactions involving YPD1 mutants . With respect to phosphoryl transfer from SLN1-R1 approximately P, only one YPD1 mutant (K67A) exhibited an increase in K(d) and thus affects binding of YPD1 to SLN1-R1 approximately P, whereas other mutants (R90A, Q86A, and G68Q) showed a decrease in phosphoryl transfer rate . Only the G68Q-YPD1 mutant was significantly affected in phosphotransfer to SSK1-R2 ( approximately 680-fold decrease in rate in comparison to wild-type) . This is the first report of a kinetic analysis of a eukaryotic "two-component" histidine-aspartic acid phosphotransfer system, enabling a comparison of the transfer rates and binding constants to the few bacterial systems that have been studied this way.

Proteomics . 2005 Jan 3; {Epub ahead of print}
Peeling the yeast protein network; Wuchty S et al.; A set of highly connected proteins (or hubs) plays an important role for the integrity of the protein interaction network of Saccharomyces cerevisae by connecting the network's intrinsic modules {1, 2} . The importance of the hubs' central placement is further confirmed by their propensity to be lethal . However, although highly emphasized, little is known about the topological coherence among the hubs . Applying a core decomposition method which allows us to identify the inherent layer structure of the protein interaction network, we find that the probability of nodes both being essential and evolutionary conserved successively increases toward the innermost cores . While connectivity alone is often not a sufficient criterion to assess a protein's functional, evolutionary and topological relevance, we classify nodes as globally and locally central depending on their appearance in the inner or outer cores . The observation that globally central proteins participate in a substantial number of protein complexes which display an elevated degree of evolutionary conservation allows us to hypothesize that globally central proteins serve as the evolutionary backbone of the proteome . Even though protein interaction data are extensively flawed, we find that our results are very robust against inaccurately determined protein interactions.

Biochemistry (Mosc), 2004 Dec, 69(12), 1391 - 8
Regulation of NAD(+)-Dependent Isocitrate Dehydrogenase in the Citrate Producing Yeast Yarrowia lipolytica; Morgunov IG et al.; The mechanism of the increased accumulation (overproduction) of citric acids in the yeast Yarrowia lipolytica while growing in the presence of glucose under nitrogen deficiency was investigated . The limitation of the yeast growth by the source of nitrogen decreases the total content of nucleotides and increases the ratios of ATP/AMP and NADH/NAD+ . NAD+-Dependent isocitrate dehydrogenase, an enzyme of the tricarboxylic acid cycle playing a key role in the regulation of biosynthesis of citric and isocitric acids, was isolated from Y . lipolytica . The molecular weights of the native enzyme and its subunits were found to be 412 and 52 kD, respectively . It is concluded that the enzyme is a homooligomer consisting of eight subunits . Investigation of the effect of some intermediates of the tricarboxylic acid cycle on the activity of this enzyme suggests that the enhanced excretion of citric acids can be caused by the inhibition of NAD+-dependent isocitrate dehydrogenase due to the decrease in the content of AMP and increase in the NADH/NAD+ ratio in the cells of Y . lipolytica under depletion of nitrogen.

Biochemistry (Mosc), 2004 Nov, 69(11), 1310 - 7
A New Alkalitolerant Yarrowia lipolytica Yeast Strain Is a Promising Model for Dissecting Properties and Regulation of Na(+)-Dependent Phosphate Transport Systems; Zvyagilskaya RA et al.; A newly isolated osmo-, salt-, and alkalitolerant Yarrowia lipolytica yeast strain is distinguished from other yeast species by its capacity to grow vigorously at alkaline pH values (9.7), which makes it a promising model organism for studying Na+-dependent phosphate transport systems in yeasts . Phosphate uptake by Y . lipolytica cells grown at pH 9.7 was mediated by several kinetically discrete Na+-dependent systems specifically activated by Na+ . One of these, a low-affinity transporter, operated at high concentrations of extracellular phosphate . The other two, high-affinity systems, maximally active in phosphate-starved cells, were repressed or derepressed depending on the prevailing extracellular phosphate concentration and pH value . The contribution of Na+/P(i)-cotransport systems to the total cellular phosphate uptake progressively increased with increasing pH, reaching its maximum at pH >/= 9.

Pest Manag Sci . 2004 Dec 31; {Epub ahead of print}
Expression of a wheat cytochrome P450 monooxygenase in yeast and its inhibition by glyphosate; Xiang WS et al.; Glyphosate is a non-selective herbicide which acts by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase . Wheat cytochrome P450 monooxygenase specifically catalyzes the metabolism of some sulfonylurea herbicides such as chlorsulfuron and triasulfuron . Here we report that glyphosate is an inhibitor of a wheat cytochrome (CYP71C6v1), the cDNA of which was amplified by RT-PCR and heterologously expressed in yeast . The microsomal fractions derived from this strain had a Soret peak at 502 nm in the reduced carbon monoxide difference spectrum, which is a typical spectral characteristic . The addition of glyphosate to the microsomal protein resulted in a Type II spectrum indicative of binding via the nitrogen group to haem of cytochrome P450 as a sixth ligand . A spectral dissociation constant, K(s) of 70 micromol litre(-1) was observed and an IC(50) of 11 micromol litre(-1) was found for glyphosate inhibition of CYP71C6v1 P450 activity . Copyright (c) 2005 Society of Chemical Industry.

Anal Chem, 2005 Jan 1, 77(1), 344 - 9
Determination of methionine and selenomethionine in selenium-enriched yeast by species-specific isotope dilution with liquid chromatography-mass spectrometry and inductively coupled plasma mass spectrometry detection; McSheehy S et al.; Selenomethionine (SeMet) and methionine (Met), liberated by acid hydrolysis of selenium-enriched yeast, were quantified by liquid chromatography-mass spectrometry (LC/MS) using standard additions calibrations as well as isotope dilution (ID) based on species-specific (13)C-enriched spikes . LC inductively coupled plasma mass spectrometry (ICPMS) was also employed for the quantification of SeMet, and (74)Se-enriched SeMet was used for ID calibration . The results were evaluated to ascertain the feasibility of using these methods in a campaign to certify selenized yeast . Good agreement was found between the methods, which, when averaged, gave concentrations of 5482.2 +/- 101 and 3256.9 +/- 217.4 microg/g for Met and SeMet, respectively . This corresponds to a 1.68:1 Met-to-SeMet ratio in the yeast . Quantification by ID LC/MS and LC ICPMS yields the most precise sets of results with relative standard deviations in the range 0.5-1.3% (n = 6) . A total selenium concentration of 2064.6 +/- 45.4 microg/g was obtained for this yeast material . The extraction efficiency and a mass balance budget were determined . Acid hydrolysis liberated 81.0% of the total selenium present . SeMet comprised 79.0% of the extracted selenium and 63.9% of the total selenium present in the yeast.

Sci China C Life Sci, 2004 Oct, 47(5), 442 - 8
Androgen receptor coregulator ARA267-alpha interacts with death receptor-6 revealed by the yeast two-hybrid; Mai T et al.; ARA267-alpha is a newly identified androgen receptor coactivator . In order to further elucidate its precise role in cells, using the ARA267-alpha fragment containing four PHD and one SET conserved domains as bait we revealed an ARA267-alpha-PHD-SET-interacting protein, death receptor-6 (DR6), in the yeast two-hybrid screening . DR6 is the member of TNF receptor family and has a death domain in its intracellular cytoplasmic portion (DR6cp) to mediate the cell apoptosis . The interaction between ARA267-alpha-PHD-SET and DR6cp was confirmed in vitro and in vivo . Our finding implied that androgen signaling pathway might cross talk with apoptosis signaling pathway through the interaction between ARA267-alpha and DR6.

Free Radic Res, 2004 Sep, 38(9), 1019 - 24
Ascorbate restores lifespan of superoxide-dismutase deficient yeast; Krzepilko A et al.; Yeast (Saccharomyces cerevisiae) mutants lacking CuZn-superoxide dismutase (CuZnSOD) are hypersensitive to oxygen and have significantly decreased replicative life span . Both these defects can be ameliorated by exogenous ascorbate . The effect of ascorbate on life span is complicated by auto-oxidation of its compound in the medium . If negative effects of auto-oxidation are prevented by exchange of the medium, ascorbate prolongs not only mean but also maximal replicative life span of the yeast in the atmosphere of air and of pure oxygen . These results demonstrate that life span shortening due to the lack of a vital antioxidant enzyme can be ameliorated by a low-molecular weight antioxidant.

Free Radic Res, 2004 Nov, 38(11), 1159 - 65
Limited effectiveness of antioxidants in the protection of yeast defective in antioxidant proteins; Lewinska A et al.; Efficacy of several antioxidants in the protection of the yeast Saccharomyces cerevisiae mutants deficient in CuZnSOD and deficient in glutaredoxin 5 to growth restriction induced by oxidants was studied . Ascorbate and glutathione protected the Deltasod1 and Deltagrx5 mutants against the effects of t-butyl hydroperoxide and cumene hydroperoxide, Deltasod1 mutants against oxytetracycline and Deltagrx5 mutants against menadione and 2,2'-azobis-(2-amidinopropane) . However, Tempol, Trolox and melatonin were much less effective, showing prooxidative effects and, at high concentrations, hampering the growth of the mutants in the absence of exogenous oxidants . These results point to a complication of cellular effects of antioxidants by their prooxidative effects and to the usefulness of cellular tests to evaluate the biological effectiveness of antioxidants.

Mech Ageing Dev, 2005 Feb, 126(2), 243 - 8
Yeast longevity and aging-the mitochondrial connection; Jazwinski SM; Studies of the yeast Saccharomyces cerevisiae reveal four processes determining life span: metabolism, stress resistance, chromatin-dependent gene regulation, and genome stability . The retrograde response, which signals mitochondrial dysfunction resulting in changes in nuclear gene expression, extends yeast life span and is induced during normal aging . This response involves extensive metabolic adaptations . The retrograde response links metabolism and genome stability during yeast aging . A reduction in the availability of nutrients also extends yeast life span . This metabolic mechanism operates by pathways distinct from the retrograde response, although it shares with the latter some longevity effectors . Life extension by calorie restriction entails re-modeling of mitochondrial function . The retrograde response appears to compensate for age changes, while calorie restriction may be a preventive mechanism . The maintenance of age asymmetry between the mother and daughter yeast cells also depends on mitochondrial function . Loss of this age asymmetry occurs during normal yeast aging and may be a paradigm for stem cell aging . The importance of mitochondrial integrity in yeast longevity is emphasized by the role of prohibition function in attenuating oxidative damage . Our studies point to the central role of mitochondria in yeast aging . They highlight the importance of the maintenance of mitochondrial membrane potential, which drives the transport of biosynthetic precursors derived from the Krebs cycle . Common threads weave their way through the studies of aging in yeast and in other model organisms . This suggests conserved features of aging across phyla.

Curr Biol, 2004 Dec 29, 14(24), 2183 - 96
Spo13 facilitates monopolin recruitment to kinetochores and regulates maintenance of centromeric cohesion during yeast meiosis; Katis VL et al.; BACKGROUND: Cells undergoing meiosis perform two consecutive divisions after a single round of DNA replication . During the first meiotic division, homologous chromosomes segregate to opposite poles . This is achieved by (1) the pairing of maternal and paternal chromosomes via recombination producing chiasmata, (2) coorientation of homologous chromosomes such that sister chromatids attach to the same spindle pole, and (3) resolution of chiasmata by proteolytic cleavage by separase of the meiotic-specific cohesin Rec8 along chromosome arms . Crucially, cohesin at centromeres is retained to allow sister centromeres to biorient at the second division . Little is known about how these meiosis I-specific events are regulated . RESULTS: Here, we show that Spo13, a centromere-associated protein produced exclusively during meiosis I, is required to prevent sister kinetochore biorientation by facilitating the recruitment of the monopolin complex to kinetochores . Spo13 is also required for the reaccumulation of securin, the persistence of centromeric cohesin during meiosis II, and the maintenance of a metaphase I arrest induced by downregulation of the APC/C activator CDC20 . CONCLUSION: Spo13 is a key regulator of several meiosis I events . The presence of Spo13 at centromere-surrounding regions is consistent with the notion that it plays a direct role in both monopolin recruitment to centromeres during meiosis I and maintenance of centromeric cohesion between the meiotic divisions . Spo13 may also limit separase activity after the first division by ensuring securin reaccumulation and, in doing so, preventing precocious removal from chromatin of centromeric cohesin.

Biochim Biophys Acta, 2005 Jan 7, 1706(1-2), 117 - 25
The yeast cAMP protein kinase Tpk3p is involved in the regulation of mitochondrial enzymatic content during growth; Chevtzoff C et al.; During aerobic cell growth, mitochondria must meet energy demand either by adjusting cellular mitochondrial content or by adjusting ATP production flux, allowing a constant growth yield . On respiratory substrate, the Ras/cAMP pathway has been shown to be involved in this process in the yeast Saccharomyces cerevisiae . We show that of the three cAMP protein kinase catalytic subunits, Tpk3p is the one specifically involved in the regulation of cellular mitochondrial content when energy demand decreases . In decreased energy demand, the Deltatpk3 mitochondrial enzymatic content decreases leading to a subsequent decrease in the cellular growth rate . Moreover, enzymatic content decreases in the Deltatpk3 isolated mitochondria, suggesting that the amount of cellular mitochondria is not affected, but rather that the mitochondria are modified . Our study points to an important decrease in the cytochrome c content in the Deltatpk3 mitochondria, which leads to a decrease in the slipping process at the level of cytochrome-c-oxidase.

Cell, 2004 Dec 29, 119(7), 991 - 1000
Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast; Bao MZ et al.; The yeast MAPK pathways required for mating versus filamentous growth share multiple components yet specify distinct programs . The mating-specific MAPK, Fus3, prevents crosstalk between the two pathways by unknown mechanisms . Here we show that pheromone signaling induces Fus3-dependent degradation of Tec1, the transcription factor specific to the filamentation pathway . Degradation requires Fus3 kinase activity and a MAPK phosphorylation site in Tec1 at threonine 273 . Fus3 associates with Tec1 in unstimulated cells, and active Fus3 phosphorylates Tec1 on T273 in vitro . Destruction of Tec1 requires the F box protein Dia2 (Digs-into-agar-2), and Cdc53, the Cullin of SCF (Skp1-Cdc53-F box) ubiquitin ligases . Notably, mutation of the phosphoacceptor site in Tec1, deletion of FUS3, or deletion of DIA2 results in a loss of signaling specificity such that pheromone pathway signaling erroneously activates filamentation pathway gene expression and invasive growth . Signal-induced destruction of a transcription factor for a competing pathway provides a mechanism for signaling specificity.

Cell, 2004 Dec 29, 119(7), 981 - 90
Fus3-regulated Tec1 degradation through SCFCdc4 determines MAPK signaling specificity during mating in yeast; Chou S et al.; Signaling specificity is fundamental for parallel mitogen-activated protein kinase (MAPK) cascades that control growth and differentiation in response to different stimuli . In Saccharomyces cerevisiae, components of the pheromone-responsive MAPK cascade activate Fus3 and Kss1 MAPKs to induce mating and Kss1 to promote filamentation . Active Fus3 is required to prevent the activation of the filamentation program during pheromone response . How Fus3 prevents the crossactivation is not clear . Here we show that Tec1, a cofactor of Ste12 for the expression of filamentation genes, is rapidly degraded during pheromone response . Fus3 but not Kss1 induces Tec1 ubiquination and degradation through the SCFCdc4 ubiquitin ligase . T273 in a predicted high-affinity Cdc4 binding motif is phosphorylated by Fus3 both in vitro and in vivo . Tec1T273V blocks Tec1 ubiquitination and degradation and allows the induction of filamentation genes in response to pheromone . Thus, Fus3 inhibits filamentous growth during mating by degrading Tec1.

J Biol Chem . 2004 Dec 24; {Epub ahead of print}
Physical and genetic interactions link the yeast protein Zds1p with mRNA nuclear export; Estruch F et al.; Eukaryotic gene expression requires the export of mRNA from nucleus to the cytoplasm . The DEAD box protein Dbp5p is an essential export factor conserved from yeast to man . A fraction of Dbp5p forms a complex with nucleoporins of the cytoplasmic filaments of the nuclear pore complex (NPC) . Gfd1p was identified originally as a multicopy suppressor of the rat8-2 ts allele of DBP5 . Here we report that Dbp5p and Gfd1p interact with Zds1p, a protein previously identified as a multicopy suppressor in several yeast genetic screens . Using the 2-hybrid system we show that Zds1p interacts in vivo with both Gfd1p and Dbp5p . In vitro binding experiments reveal that Gfd1p and Dbp5p bind directly to the C-terminal part of Zds1p . In addition, ZDS1 interacts genetically with mutant alleles of genes encoding key factors in mRNA export, including DBP5 and MEX67 . Furthermore, deletion of ZDS1 or of both ZDS1 and the closely-related ZDS2 exacerbates the poly(A)+ export defects shown by dbp5-2 and mex67-5 mutants . We propose that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the NPC and is required for optimal mRNA export.

Drug Metab Pharmacokinet, 2002, 17(2), 117 - 24
Molecular Cloning of Monkey CYP2C43 cDNA and Expression in Yeast; Matsunaga T et al.; A cDNA clone designated as CYP2C43 was isolated from the rhesus monkey liver cDNA library . The first 16 amino acid residues at the N-terminal region of this cDNA product were identical with those of P450 CMLd which have been purified and characterized as S-mephenytoin 4'-hydroxylase in monkey liver . The respective nucleotide and deduced amino acid sequences of CYP2C43 were 83% and 77%, identical to those of monkey CYP2C20 . Antibody against CYP2C9 detected a protein in the microsomes of yeast transformed CYP2C43 expression plasmid . The specific content of recombinant CYP2C43 was 78.0 pmol/mg protein and the yield was 4.23 nmol/l of the culture . CYP2C43 was able to metabolize S-mephenytoin stereo-selectively . The activity for S-mephenytoin in the microsomes reconstituted with or without cytochrome b(5) was found to be 96.2 or 23.7 pmol/min/nmol P450, respectively . CYP2C43, however, did not show any oxidative activity for tolbutamide . These results indicate that CYP2C43 is the second identified member of the monkey CYP2C subfamily and a cDNA clone encoding P450 CMLd in monkey.

Plant Physiol, 2005 Jan, 137(1), 308 - 16 Epub 2004 Dec 23.
The Cytokinin Requirement for Cell Division in Cultured Nicotiana plumbaginifolia Cells Can Be Satisfied by Yeast Cdc25 Protein Tyrosine Phosphatase . Implications for Mechanisms of Cytokinin Response and Plant Development; Zhang K et al.; Cultured cells of Nicotiana plumbaginifolia, when deprived of exogenous cytokinin, arrest in G2 phase prior to mitosis and then contain cyclin-dependent protein kinase (CDK) that is inactive because phosphorylated on tyrosine (Tyr) . The action of cytokinin in stimulating the activation of CDK by removal of inhibitory phosphorylation from Tyr is not a secondary downstream consequence of other hormone actions but is the key primary effect of the hormone in its stimulation of cell proliferation, since cytokinin could be replaced by expression of cdc25, which encodes the main Cdc2 (CDK)-Tyr dephosphorylating enzyme of yeast (Saccharomyces cerevisiae) . The cdc25 gene, under control of a steroid-inducible promoter, induced a rise in cdc25 mRNA, accumulation of p67(Cdc25) protein, and increase in Cdc25 phosphatase activity that was measured in vitro with Tyr-phosphorylated Cdc2 as substrate . Cdc25 phosphatase activity peaked during mitotic prophase at the time CDK activation was most rapid . Mitosis that was induced by cytokinin also involved increase in endogenous plant CDK Tyr phosphatase activity during prophase, therefore indicating that this is a normal part of plant mitosis . These results suggest a biochemical mechanism for several previously described transgene phenotypes in whole plants and suggest that a primary signal from cytokinin leading to progression through mitosis is the activation of CDK by dephosphorylation of Tyr.

Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 163 - 8 Epub 2004 Dec 23.
The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function; Huang HR et al.; Group I and II introns self-splice in vitro, but require proteins for efficient splicing in vivo, to stabilize the catalytically active RNA structure . Recent studies showed that the splicing of some Neurospora crassa mitochondrial group I introns additionally requires a DEAD-box protein, CYT-19, which acts as an RNA chaperone to resolve nonnative structures formed during RNA folding . Here we show that, in Saccharomyces cerevisiae mitochondria, a related DEAD-box protein, Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs, and that all these defects can be partially or completely suppressed by the expression of CYT-19 . Results for the aI2 group II intron indicate that Mss116p is needed after binding the intron-encoded maturase, likely for the disruption of stable but inactive RNA structures . Our results suggest that both group I and II introns are prone to kinetic traps in RNA folding in vivo and that the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones.

J Biol Chem . 2004 Dec 23; {Epub ahead of print}
Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids; Salnikova AB et al.; The {PSI+} prion determinant of Saccharomyces cerevisiae causes nonsense suppressor phenotype due to a reduced function of the translation termination factor Sup35 (eRF3) polymerized into amyloid fibrils . Prion state of the Rnq1 protein, {PIN+}, is required for the {PSI+} de novo generation, but not propagation . Yeast {psi-} {PIN+} cells overproducing Sup35 can exhibit nonsense suppression without generation of a stable {PSI+} . Here we show that in such cells most of Sup35 represents amyloid polymers, though remaining Sup35 monomer is sufficient for normal translation termination . Presence of these polymers strictly depends on {PIN+}, suggesting that their maintenance relies on efficient generation de novo, rather than inheritance . Sup35 polymers contain Rnq1, confirming a hypothesis that Rnq1 polymers seed Sup35 polymerization . About 10 percents of cells overproducing Sup35 form colonies on medium selective for suppression which suggests that the proportion of Sup35 monomers to polymers varies between cells of transformants allowing selection of cells deficient for soluble Sup35 . A hybrid Sup35 with the N-terminal domain replaced for 66 glutamine residues also polymerizes and can cause nonsense suppression when overproduced . The described polymers of these proteins differ from the {PSI+} polymers by poor heritability and very high frequency of the de novo appearance, thus being more similar to amyloids than to prions.

Biophys Chem, 2005 Jan 1, 113(1), 41 - 51
Single-molecule detection of yeast cytochrome c by Surface-Enhanced Raman Spectroscopy; Delfino I et al.; The giant enhancement of Raman signal near silver colloidal nanoparticles is exploited to study the Raman spectrum of Cytochrome c from Saccharomyces cerevisiae (Yeast Cytochrome c-YCc) in the limit of single-molecule . The investigation is performed on proteins both in solution and immobilised onto a glass slide using a quasi resonant laser line as exciting source with low excitation intensity . In both cases, spectra acquired at different times exhibit dramatic temporal fluctuations in both the total spectrum and in the specific line intensity, even though averaging of several individual spectra reproduces the main Raman features of bulk YCc . Analysis of the spectral intensity fluctuations from solutions reveals a multimodal distribution of some specific Raman lines, consistent with the approaching of single molecule regime . Among other results, the statistical analysis of the spectra from immobilised samples seems to indicate dynamical processes involving the reorientational of the heme with respect to the metal surface.

EMBO J . 2004 Dec 16; {Epub ahead of print}
A novel gene amplification system in yeast based on double rolling-circle replication; Watanabe T et al.; Gene amplification is involved in various biological phenomena such as cancer development and drug resistance . However, the mechanism is largely unknown because of the complexity of the amplification process . We describe a gene amplification system in Saccharomyces cerevisiae that is based on double rolling-circle replication utilizing break-induced replication . This system produced three types of amplification products . Type-1 products contain 5-7 inverted copies of the amplification marker, leu2d . Type-2 products contain 13 to approximately 100 copies of leu2d (up to approximately 730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2d copies . Type-3 products are acentric multicopy minichromosomes carrying leu2d . Structures of type-2 and -3 products resemble those of homogeneously staining region and double minutes of higher eukaryotes, respectively . Interestingly, products analogous to these were generated at low frequency without deliberate DNA cleavage . These features strongly suggest that the processes described here may contribute to natural gene amplification in higher eukaryotes.

Nature, 2004 Dec 23, 432(7020), 1054 - 8
The transcription factor Ifh1 is a key regulator of yeast ribosomal protein genes; Wade JT et al.; Ribosomal protein (RP) genes in eukaryotes are coordinately regulated in response to growth stimuli and environmental stress, thereby permitting cells to adjust ribosome number and overall protein synthetic capacity to physiological conditions . Approximately 50% of RNA polymerase II transcription is devoted to RP genes . The transcriptional regulator Rap1 binds most yeast RP promoters, and Rap1 sites are important for coordinate regulation of RP genes . However, Rap1 is not the specific regulator that controls RP transcription because it also functions as a repressor, and many Rap1-activated promoters are not coordinately regulated with RP promoters . Here we show that the transcription factors Fhl1 and Ifh1 associate almost exclusively with RP promoters; association depends on Rap1 and (to a lesser extent) a DNA element at many RP promoters . Ifh1 is recruited to promoters via the forkhead-associated (FHA) domain of Fhl1; the level of Ifh1 associated with RP promoters determines the level of transcription; and environmental stress causes a marked reduction in the association of Ifh1, but not Fhl1 or Rap1 . Thus, Ifh1 association with promoters is the key regulatory step for coordinate expression of RP genes.

Analyst, 2005 Jan, 130(1), 35 - 7 Epub 2004 Nov 22.
Identification of selenomethionine in selenized yeast using two-dimensional liquid chromatography-mass spectrometry based proteomic analysis; McSheehy S et al.; Selenium-enriched yeast has been commonly used as a nutritional supplement . Here we describe a protocol used to investigate the metabolic fate of inorganic selenium in yeast . We provide definitive, mass spectrometry based evidence for the non-specific incorporation of selenomethionine in the yeast proteome involving the replacement of about 30% of all methionine with selenomethionine.

RNA . 2004 Dec 21; {Epub ahead of print}
RNA-protein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening; Hook B et al.; The yeast three-hybrid system has become a useful tool in analyzing RNA-protein interactions . An RNA sequence is tested in combination with an RNA-binding protein linked to a transcription activation domain (AD) . A productive RNA-protein interaction activates a reporter gene in vivo . The system has been used to test candidate RNA-protein pairs, to isolate mutations in each interacting partner, and to identify proteins that bind a given RNA sequence . However, the relationship between reporter gene activation and in vitro affinity of an RNA-protein interaction has not been examined systematically . This limits interpretation of the data and complicates the development of new strategies . Here, we analyze several key parameters of the three-hybrid system, using as a model the interaction of a PUF protein, FBF-1, with a range of RNA targets . We compare activation of two reporter genes as a function of the in vitro affinity of the interaction . HIS3 and LacZ expression levels are directly related to affinity over a 10-fold range of Kd . Expression of the reporter genes also is directly related to the abundance of the activation domain fusion protein . We describe a new yeast strain, YBZ1, that simplifies screening of cDNA/AD libraries . This strain possesses a tandem, head-to-tail dimer of a high-affinity variant of MS2 coat protein, fused to a monomer of the LexA DNA-binding protein . We show that the use of this strain in cDNA library screens increases the number of genuine, sequence-specific positives detected, and at the same time reduces the background of false, RNA-independent positives.

RNA . 2004 Dec 21; {Epub ahead of print}
Nonsense-associated alternative splicing of T-cell receptor {beta} genes: No evidence for frame dependence; Mohn F et al.; Mutations that generate premature translation-termination codons (PTCs) often result in production of alternatively spliced mRNAs . While in many cases, the PTC-causing mutation was found to affect splicing directly by disrupting an exonic splicing enhancer, induction of alternative splicing of TCR-betapre-mRNA has been reported to be specific for mutations that prematurely terminate the open reading frame . During testing of a cyto-nuclear feedback model that would have explained how cytoplasmic translation could influence nuclear splicing of TCR-betatranscripts, control experiments questioned the frame dependence of the nonsense-associated altered splicing (NAS) of TCR-betapre-mRNA . A subsequent detailed analysis of alternatively spliced TCR-betamRNA expressed from different minigene constructs with nonsense, silent, or frame-shift mutations at various positions revealed no correlation between truncation of the reading frame and production of alternatively spliced mRNA . Our study thus contradicts the previously reported PTC specificity of TCR-betaNAS and points out the need for systematically testing the PTC specificity in other cases where NAS has been observed.

Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 51 - 6 Epub 2004 Dec 21.
The crystal structure of yeast copper thionein: the solution of a long-lasting enigma; Calderone V et al.; We report here the crystal structure of yeast copper thionein (Cu-MT), determined at 1.44-A resolution . The Cu-MT structure shows the largest known oligonuclear Cu(I) thiolate cluster in biology, consisting of six trigonally and two digonally coordinated Cu(I) ions . This is at variance with the results from previous spectroscopic determinations, which were performed on MT samples containing seven rather than eight metal ions . The protein backbone has a random coil structure with the loops enfolding the copper cluster, which is located in a cleft where it is bound to 10 cysteine residues . The protein structure is somewhat different from that of Ag(7)-MT and similar, but not identical, to that of Cu(7)-MT . Besides the different structure of the metal cluster, the main differences lie in the cysteine topology and in the conformation of some portions of the backbone . The present structure suggests that Cu-MT, in addition to its role as a safe depository for copper ions in the cell, may play an active role in the delivery of copper to metal-free chaperones.

Syst Appl Microbiol, 2004 Nov, 27(6), 661 - 71
Trichosporon mycotoxinivorans sp . nov., a new yeast species useful in biological detoxification of various mycotoxins; Molnar O et al.; A yeast strain isolated from the hindgut of the lower termite Mastotermes darwiniensis (Mastotermitidae) was found to represent a new member of the genus Trichosporon . Trichosporon mycotoxinivorans is closely related to T . loubieri on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA, an approx . 600 bp fragment of the 18S rDNA and both ITS regions . However, the two species differ at nine positions in the D1/D2 region of 26S rDNA . The IGS1 region of T . mycotoxinivorans is 401 bp long . T . mycotoxinivorans is distinguished from T . loubieri by its ability to assimilate inulin and galactitol, and its inability to grow at 40 degrees C . The name of this newly isolated strain refers to an important characteristics of T . mycotoxinivorans to detoxify mycotoxins such as ochratoxin A and zearalenone . Therefore this strain can be used for the deactivation of the respective mycotoxins in animal feeds.

Wei Sheng Yan Jiu, 2004 Sep, 33(5), 565 - 9
{Safety assessment of GM yeast feed additive with cecropin CAD gene}; Deng P et al.; OBJECTIVE: To evaluate the safety of GM yeast feed additive with cecropin CAD and to study and set up a model of Safety assessment for GM feed and detecting method . METHODS: To ensure the safety of the GM products, it has been done that to detect and value the safety of receptor organisms and expression products of extrinic gene, the genetic stability of biologic properties of genomic modified yeast feed and condition of transfer and cumulation of anti-bacterial peptide and its products in circumstance and the feeded animals . RESULT AND CONCLUSION: The receptor animals and expression products of extrinic gene are safe, and the genomic modified products have steady genetic characters . The cectopin CAD neither cumulates in feeded animal nor releases into environment . The genomic modified feed additive is safe.

Cell Cycle . 2005 Jan 29;4(1) {Epub ahead of print}
A Transcriptional Pathway for Cell Separation in Fission Yeast; Bahler J; Numerous genes are transcriptionally activated and repressed in a cell cycle-dependent manner . We have recently reported the global gene expression program during the cell cycle in fission yeast (S . pombe) . Among the periodically expressed fission yeast genes, a large proportion shows peak transcript levels during mitosis . Many of these genes are regulated by a transcriptional cascade involving two transcription factors: the forkhead protein Sep1p which activates the zinc finger protein Ace2p . A main function of the Sep1p-Ace2p transcriptional pathway is to trigger the separation of daughter cells after cytokinesis . Absence of Sep1p, Ace2p, or some of their target genes leads to a hyphal-like growth pattern with chains of connected cells . Yeast cells probably evolved from filamentous fungi . It is possible that the Sep1p-Ace2p pathway contributed to the emergence of proliferation through single cells, and that this regulatory pathway can still be modulated to adjust growth modes depending on environmental conditions . Here, various properties of the Sep1p-Ace2p transcriptional pathway and mechanisms for cell separation are discussed in the context of recent findings.

Genetics, 2004 Dec, 168(4), 2421 - 4
Does chromosome size affect map distance and genetic interference in budding yeast?
Turney D, de Los Santos T, Hollingsworth NM.
The hypothesis that chromosome size affects the rate and distribution of meiotic crossovers in budding yeast was tested . Map distance and interference were measured in the same genetic intervals present on either small (340 and 508 kb) or large (917 and 1085 kb) chromosomes, respectively . No differences were observed.

Genetics, 2004 Dec, 168(4), 1827 - 41
Genetic analysis of yeast yip1p function reveals a requirement for Golgi-localized rab proteins and rab-Guanine nucleotide dissociation inhibitor; Chen CZ et al.; Yip1p is the first identified Rab-interacting membrane protein and the founder member of the YIP1 family, with both orthologs and paralogs found in all eukaryotic genomes . The exact role of Yip1p is unclear; YIP1 is an essential gene and defective alleles severely disrupt membrane transport and inhibit ER vesicle budding . Yip1p has the ability to physically interact with Rab proteins and the nature of this interaction has led to suggestions that Yip1p may function in the process by which Rab proteins translocate between cytosol and membranes . In this study we have investigated the physiological requirements for Yip1p action . Yip1p function requires Rab-GDI and Rab proteins, and several mutations that abrogate Yip1p function lack Rab-interacting capability . We have previously shown that Yip1p in detergent extracts has the capability to physically interact with Rab proteins in a promiscuous manner; however, a genetic analysis that covers every yeast Rab reveals that the Rab requirement in vivo is exclusively confined to a subset of Rab proteins that are localized to the Golgi apparatus.

J Biol Chem . 2004 Dec 17; {Epub ahead of print}
Conditional osmotic stress in yeast: A system to study transport through aquaglyceroporins and osmostress signaling; Karlgren S et al.; The accumulation and transport of solutes are hallmarks of osmoadaptation . In this study we have employed the inability of the Saccharomyces cerevisiae gpd1 gpd2 mutant both to produce glycerol and to adapt to high osmolarity in order to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling . High levels of different polyols, including glycerol, inhibited growth of the gpd1 gpd2 mutant . This growth inhibition was suppressed by expression of the hyperactive allele Fps1-1 of the osmogated yeast aquaglyceroporin, Fps1 . The degree of suppression correlated with the relative rate of transport of the different polyols tested . Transport studies in secretory vesicles confirmed that Fps1-1 transports polyols at increased rates compared with wild type Fps1 . Importantly, wild type Fps1 and Fps1-1 showed similarly low permeability for water . The growth defect on polyols in the gpd1 gpd2 mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9 . We surmised that this suppression was due to polyol influx causing the cells to passively adapt to the stress . Indeed, when aquaglyceroporin-expressing gpd1 gpd2 mutants were treated with glycerol, xylitol or sorbitol the osmosensing HOG pathway was activated and the period of activation correlated with the apparent rate of polyol uptake . This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation . Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.

J Biol Chem . 2004 Dec 20; {Epub ahead of print}
Yeast flavohemoglobin, a nitric oxide oxidoreductase, is located both in the cytosol and mitochondrial matrix; Cassanova N et al.; Yeast flavohemoglobin, YHb, encoded by the nuclear gene, YHB1, has been implicated in both the oxidative and nitrosidative stress responses in Saccharomyces cerevisiae . Previous studies have shown that expression of YHB1 is optimal in normoxic or hyperoxic conditions yet respiring yeast cells have low levels of reduced YHb pigment, detected by carbon monoxide (CO) photolysis difference spectroscopy of glucose-reduced cells . Here, we have addressed this apparent discrepancy by determining the intracellular location of the YHb protein and analyzing the relationships among respiration, YHb level, and intracellular location . We have found that although intact respiration-proficient cells lack a YHb CO-spectral signature, cell extracts from these cells have both a YHb CO-spectral signature and nitric oxide (NO) consuming activity . This suggests either that YHb can not be reduced in vivo or that YHb heme is maintained in an oxidized state in respiring cells . By using an anti-YHb antibody, CO-difference spectroscopy, and by measuring NO consumption we have found that YHb localizes to two distinct intracellular compartments, the mitochondrial matrix and the cytosol, in respiring cells . Moreover, we have found that the distribution of YHb between these two compartments is affected by the presence or absence of oxygen and by the mitochondrial genome . These findings suggest that YHb functions in oxidative stress indirectly by consuming NO, which inhibits mitochondrial respiration and leads to enhanced production of reactive oxygen species, and that cells can regulate intracellular distribution of YHb in accordance with this function.

J Biol Chem . 2004 Dec 20; {Epub ahead of print}
Genome wide analysis reveals inositol, not choline, as the major effector of Ino2p-Ino4p and unfolded protein response target gene expression in yeast; Jesch SA et al.; In the yeast Saccharomyces cerevisiae the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline . A genome-wide approach using cDNA microarray technology was utilized to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline . We report that the global response to inositol is completely distinct from the effect of choline . Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating . Moreover, the combination inositol and choline increased the number of repressed genes compared to inositol alone and enhanced the repression levels of a subset of genes that responded to inositol . In all, 110 genes were repressed in the presence of inositol and choline . Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild type cells . One set of genes contained the UASINO sequence and were bound by Ino2p and Ino4p . Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p . Another non-overlapping set of genes were coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but were not dependent on OPI1 and did not show further repression when choline was present together with inositol . These results suggest that inositol is the major effector of target gene expression, while choline plays a minor role.

J Biol Chem . 2004 Dec 14; {Epub ahead of print}
Native replication intermediates of the yeast 20 S RNA virus have a single-stranded RNA backbone; Fujimura T et al.; 20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae . The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo . A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome . When in vitro products, after phenol extraction, were analyzed in time course, radioactive nucleotides were first incorporated into double-stranded RNA intermediates and then chased out to the final single-stranded RNA products . The positive and negative strands in these dsRNA intermediates were non-covalently associated and the release of the positive strand products from the intermediates required a net RNA synthesis . We found, however, that these dsRNA intermediates were an artifact caused by phenol-extraction . Native replication intermediates had an ssRNA backbone as judged by RNase sensitivity experiments and they migrated distinctly from a dsRNA form in non-denaturing gels . Upon completion of RNA synthesis, positive strand RNA products as well as negative strands templates were released from replication intermediates . These results indicate that the native replication intermediates consists of a positive strand less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91 . Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 masculineC is not an intermediate in the 20 S RNA replication cycle, but a byproduct.

Mech Ageing Dev, 2005 Jan, 126(1), 17 - 21
Large-scale identification in yeast of conserved ageing genes; Kaeberlein M et al.; Recent advances have suggested the existence of phylogenetically conserved pathways regulating ageing in eukaryotes . At least two of these "public" longevity-determining pathways appear to have been evolutionarily conserved from yeast through mammals . We have developed a high-throughput, genome-wide approach to identify a large fraction of the non-essential, single-gene deletion mutations that confer increased longevity in yeast . The identification and characterization of conserved genes that regulate the ageing process across eukaryotic species is likely to result in an improved understanding of the causes of human ageing and provide potential therapeutic targets for drug discovery.

Mech Ageing Dev, 2005 Jan, 126(1), 11 - 6
Analysis of gene expression profile in yeast aging chronologically; Fabrizio P et al.; The use of simple model systems such as Saccharomyces cerevisiae and Caenorhabditis elegans has played a primary role in the identification of proteins and pathways that regulate the aging process in eukaryotes . Recent findings have shown that analogous pathways regulate aging in higher eukaryotes and suggest a conserved origin for the molecular mechanisms that regulate stress-resistance and longevity . Genomics approaches that allow the simultaneous monitoring of the expression of thousands of genes are beginning to reveal the complexity of the molecular changes required to extend life span . Here we describe how analysis of the gene expression profiles of wild-type and long-lived yeast aging chronologically can be used to identify proteins that increase stress-resistance and longevity . We also discuss a novel genomics method for the identification of chronologically long-lived yeast mutants.

Biochemistry, 2004 Dec 28, 43(51), 16254 - 62
Yeast frataxin solution structure, iron binding, and ferrochelatase interaction; He Y et al.; The mitochondrial protein frataxin is essential for cellular regulation of iron homeostasis . Although the exact function of frataxin is not yet clear, recent reports indicate the protein binds iron and can act as a mitochondrial iron chaperone to transport Fe(II) to ferrochelatase and ISU proteins within the heme and iron-sulfur cluster biosynthetic pathways, respectively . We have determined the solution structure of apo yeast frataxin to provide a structural basis of how frataxin binds and donates iron to the ferrochelatase . While the protein's alpha-beta-sandwich structural motif is similar to that observed for human and bacterial frataxins, the yeast structure presented in this report includes the full N-terminus observed for the mature processed protein found within the mitochondrion . In addition, NMR spectroscopy was used to identify frataxin amino acids that are perturbed by the presence of iron . Conserved acidic residues in the helix 1-strand 1 protein region undergo amide chemical shift changes in the presence of Fe(II), indicating a possible iron-binding site on frataxin . NMR spectroscopy was further used to identify the intermolecular binding interface between ferrochelatase and frataxin . Ferrochelatase appears to bind to frataxin's helical plane in a manner that includes its iron-binding interface.

Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D348 - 52
The Ashbya Genome Database (AGD)--a tool for the yeast community and genome biologists; Hermida L et al.; The Ashbya Genome Database (AGD) is a comprehensive online source of information covering genes from the filamentous fungus Ashbya gossypii . The database content is based upon comparative genome annotation between A.gossypii and the closely related budding yeast Saccharomyces cerevisiae taking both sequence similarity and synteny (conserved order and orientation) into account . Release 2 of AGD contains 4718 protein-encoding loci located across seven chromosomes . Information can be retrieved using systematic or standard locus names from A.gossypii as well as budding and fission yeast . Approximately 90% of the genes in the genome of A.gossypii are homologous and syntenic to loci of budding yeast . Therefore, AGD is a useful tool not only for the various yeast communities in general but also for biologists who are interested in evolutionary aspects of genome research and comparative genome annotation . The database provides scientists with a convenient graphical user interface that includes various locus search and genome browsing options, data download and export functionalities and numerous reciprocal links to external databases including SGD, MIPS, GeneDB, KEGG, GermOnline and Swiss-Prot/TrEMBL . AGD is accessible at http://agd.unibas.ch/.

Curr Genet . 2004 Dec 17; {Epub ahead of print}
Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures; Chen X et al.; The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures . Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD . ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol . The extreme mutant type degraded with a half-life of approximately 12 min, whereas the normal iso-1 was stable over hours . ADD was influenced by the rho(+)/rho(-) state and by numerous chromosomal genes . Most importantly, ADD appeared to be specifically suppressed to various extents by deletions of any of the YME1, AFG3, or RCA1 genes encoding membrane-associated mitochondrial proteases, probably because the amphipathic structures caused a stronger association with the mitochondrial inner membrane and its associated proteases . The use of ADD assisted in the differentiation of substrates of different mitochondrial degradation pathways.

Plant Mol Biol, 2004 Sep, 56(1), 91 - 110
Temporally regulated expression of a yeast invertase in potato tubers allows dissection of the complex metabolic phenotype obtained following its constitutive expression; Junker BH et al.; The constitutive cytosolic expression of a yeast ( Saccharomyces cerevisiae ) invertase within potato ( Solanum tuberosum ) tubers has previously been documented to produce a dramatic metabolic phenotype in which glycolysis, respiration and amino acid synthesis are markedly enhanced at the cost of starch synthesis . These transgenic lines were further characterised by a massive cycle of sucrose degradation and resynthesis via sucrose-phosphate synthase . We have recently developed a B33 patatin driven alc gene construct allowing tight chemical control of gene expression following supply of acetaldehyde with minimal pleiotropic effects of the inducing agent on metabolism . This construct was used for chemical induction of the yeast invertase gene after 10-weeks growth to dissect the complex metabolic phenotype obtained after constitute expression . Inducible expression led to increased invertase activity within 24 h in well-defined areas within growing tubers . Although the sucrose levels were reduced, there was no effect on the levels of starch whilst levels of many amino acids decreased . Labelling experiments revealed that these lines exhibited increased rates of sucrose cycling, whereas rates of glycolysis and of starch synthesis were not substantially changed . From these results we conclude that sucrose cycling is stimulated in response to a short-term increase in the rate of sucrose mobilisation, providing evidence for a role of sucrose cycling as a buffering capacity that regulates the net rate of sucrose usage . In contrast, the dramatic increase in hexose-phosphate levels and the switch from starch synthesis to respiration seen on the constitutive expression of the invertase was not observed in the inducible lines, suggesting that this is the result of cumulative pleiotropic effects that occurred when the transgene was expressed throughout development.

Plant Mol Biol, 2004 Jul, 55(5), 645 - 62
Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis; Bender-Machado L et al.; Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions . Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated . To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants . Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels . The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments . The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate . In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.

Plant Mol Biol, 2004 Apr, 54(6), 881 - 93
Tissue-specific expression and functional complementation of a yeast potassium-uptake mutant by a salt-induced ice plant gene mcSKD1; Jou Y et al.; A full-length salt-induced transcript homologous to SKD1 (suppressor of K(+) transport growth defect) of the AAA (ATPase associated with a variety of cellular activities)-type ATPase family has been identified from the halophyte Mesembryanthemum crystallinum (ice plant) . The expression of mcSKD1 was induced by 200 mM NaCl or higher in cultured ice plant cells . When cultured ice plant cells were grown in a high K(+) (42.6 mM) medium, the level of mcSKD1 expression decreased . At the whole plant level, constitutive expression of mcSKD1 was observed in roots, stems, leaves and floral organs . Addition of 400 mM NaCl increased the transcript level in roots and stems . The expression of atSKD1 , a homologue gene in Arabidopsis , was down regulated by salt stress . Under salt stress, mcSKD1 was preferentially expressed in the outer cortex of roots and stems and in the epidermal bladder cells of leaves . The mcSKD1 transcript was constitutively expressed in placenta and integuments of the developing floral buds . Expression of the full-length or C-terminal deletion of mcSKD1 was able to complement the K(+) uptake-defect phenotype in mutant Saccharomyces cerevisiae , which is defective in high- and low-affinity K(+) uptake . Deletion of the N-terminal coiled-coil motif of mcSKD1, a structure required for membrane association, resulted in greatly reduced K(+) transport . Expression of mcSKD1 also increased the salt-tolerant ability of yeast mutants and either N- or C-terminal deletion decreased the efficiency . The physiological relevancies of mcSKD1 for K(+) uptake under high salinity environments are discussed.

Mol Cell Proteomics . 2004 Dec 22; {Epub ahead of print}
Automated yeast two hybrid screening for nuclear receptor-interacting proteins; Albers M et al.; High-throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research . Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors . A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs . We show that simple statistical parameters can be used to narrow down the dataset to a high-confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs . Within the high-confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors . We discuss several examples of high interest, and we expect that communication of this huge dataset will help to complement our knowledge of the protein-interaction repertoire of this family of transcription factors, and instigate the characterisation of the various novel candidate interactors.

Hum Genomics, 2004 Jan, 1(2), 146 - 9
The truth about mouse, human, worms and yeast; Nelson DR et al.; Genome comparisons are behind the powerful new annotation methods being developed to find all human genes, as well as genes from other genomes . Genomes are now frequently being studied in pairs to provide cross-comparison datasets . This 'Noah's Ark' approach often reveals unsuspected genes and may support the deletion of false-positive predictions . Joining mouse and human as the cross-comparison dataset for the first two mammals are: two Drosophila species, D . melanogaster and D . pseudoobscura; two sea squirts, Ciona intestinalis and Ciona savignyi; four yeast (Saccharomyces) species; two nematodes, Caenorhabditis elegans and Caenorhabditis briggsae; and two pufferfish (Takefugu rubripes and Tetraodon nigroviridis) . Even genomes like yeast and C . elegans, which have been known for more than five years, are now being significantly improved . Methods developed for yeast or nematodes will now be applied to mouse and human, and soon to additional mammals such as rat and dog, to identify all the mammalian protein-coding genes . Current large disparities between human Unigene predictions (127,835 genes) and gene-scanning methods (45,000 genes) still need to be resolved . This will be the challenge during the next few years.

J Biochem (Tokyo), 2004 Sep, 136(3), 267 - 72
Regulation of the Osmoregulatory HOG MAPK Cascade in Yeast; Saito H et al.; The budding yeast Saccharomyces cerevisiae has at least five signal pathways containing a MAP kinase (MAPK) cascade . The high osmolarity glycerol (HOG) MAPK pathway is essential for yeast survival in high osmolarity environment . This mini-review surveys recent developments in regulation of the HOG pathway with specific emphasis on the roles of protein phosphatases and protein subcellular localization . The Hog1 MAPK in the HOG pathway is negatively regulated jointly by the protein tyrosine phosphatases Ptp2/Ptp3 and the type 2 protein phosphatases Ptc1/Ptc2/Ptc3 . Specificities of these phosphatases are determined by docking interactions as well as their cellular localizations . The subcellular localizations of the osmosensors (Sln1 and Sho1), kinases (Pbs2, Hog1), and phosphatases in the HOG pathway are intricately regulated to achieve their specific functions.

Nucleic Acids Res, 2004 Dec 14, 32(22), 6469 - 78 Print 2004.
Repression of the yeast HO gene by the MATalpha2 and MATa1 homeodomain proteins; Mathias JR et al.; The HO gene in Saccharomyces cerevisiae is regulated by a large and complex promoter that is similar to promoters in higher order eukaryotes . Within this promoter are 10 potential binding sites for the a1-alpha2 heterodimer, which represses HO and other haploid-specific genes in diploid yeast cells . We have determined that a1-alpha2 binds to these sites with differing affinity, and that while certain strong-affinity sites are crucial for repression of HO, some of the weak-affinity sites are dispensable . However, these weak-affinity a1-alpha2-binding sites are strongly conserved in related yeast species and have a role in maintaining repression upon the loss of strong-affinity sites . We found that these weak sites are sufficient for a1-alpha2 to partially repress HO and recruit the Tup1-Cyc8 (Tup1-Ssn6) co-repressor complex to the HO promoter . We demonstrate that the Swi5 activator protein is not bound to URS1 in diploid cells, suggesting that recruitment of the Tup1-Cyc8 complex by a1-alpha2 prevents DNA binding by activator proteins resulting in repression of HO.

J Biol Chem . 2004 Dec 14; {Epub ahead of print}
Phosphorylation by Pho85 cyclin-dependent kinase acts as a signal for the down-regulation of the yeast sphingoid long-chain base kinase Lcb4 during the stationary phase; Iwaki S et al.; Sphingoid long-chain base 1-phosphates (LCBPs) act as bioactive lipid molecules in eukaryotic cells . In yeast, LCBPs are synthesized mainly by the long-chain base kinase Lcb4p . Until now, the regulatory mechanism for Lcb4p has been unclear . In the present study, we found that Lcb4p is post-translationally modified by phosphorylation . Using a protein kinase mutant yeast collection, we further demonstrated that the cyclin-dependent kinase Pho85p is involved in this phosphorylation . Pho85p functions in a number of cellular processes, especially in response to environmental changes . Two of ten Pho85p cyclins, Pcl1p and Pcl2p had overlapping functions in the phosphorylation of Lcb4p . Site-directed mutagenesis identified the phosphorylation sites in Lcb4p as Ser451 and Ser455 . Additionally, pulse-chase experiments revealed that Lcb4p is degraded via the ubiquitin-dependent pathway . The protein was stabilized in deltapho85 cells, suggesting that phosphorylation acts as a signal for the degradation . Lcb4p is down-regulated in the stationary phase of cell growth, and both phosphorylation and ubiquitination appear to be important for this process . Moreover, we demonstrated that Lcb4p is delivered to the vacuole for degradation via the multivesicular body . Since forced accumulation of LCBPs results in prolonged growth during the stationary phase, down-regulation of Lcb4p may be physiologically important for proper cellular responses to nutrient deprivation.

Phytother Res, 2004 Nov, 18(11), 950 - 3
Yeast hydrolysate reduces body fat of dietary obese rats; Kim KM et al.; The purpose of this study was to assess the antiobesity effect of the yeast hydrolysate (DNF) on the body weight, body fat and plasma lipids levels of high-fat fed rats . The weight gain of the HF (high fat diet) (162.58 +/- 6.68 g) was significantly (p < 0.05) greater than that of DNF-1, DNF-2, (high fat diet with DNF of 0.5 and 1.0 g/kg body weight, respectively) and control groups (143.19 +/- 7.33 g, 139.20 +/- 8.36 g, 130.23 +/- 8.02 g, respectively) . The wet weight of the epididymal fat and the perirenal fat pads of the DNF-1, DNF-2 and control groups were reduced significantly (p < 0.05) . A significant (p < 0.05) increase of HDL-cholesterol level of the DNF-2 and control groups was observed . However, there was no significant difference between DNF-1 and DNF-2 . It was also found that the triacylglycerol (TG) levels decreased significantly (p < 0.05) in the DNF-2 group from that of the HF, but there was no significant (p < 0.05) difference between DNF-1 and DNF-2 .

Mol Cell Proteomics . 2004 Dec 13; {Epub ahead of print}
Identification of sumoylated proteins by systematic immunoprecipitation of the budding yeast proteome; Wykoff DD et al.; The identification of post-translational modifications to proteins is critical for understanding many important aspects of biology . Utilizing a collection of epitope-tagged yeast strains, we developed a novel approach to determine which proteins are modified by the small ubiquitin related modifier, SUMO . We crossed traits useful for the detection of SUMO conjugation into 4246 TAP-tagged strains and successfully immunoprecipitated and screened 2893 of these proteins for association with SUMO (approximately 70% of the expressed proteome detectable by immunoblot analysis) . We found 82 proteins associated with SUMO, including many of low abundance . Because our screen was performed under non-denaturing conditions, we were able to identify multiple members of four complexes that were associated with SUMO: the RSC chromatin remodeling complex, the mediator complex, the TFIID complex, and the septin complex . In addition, we describe 5 new direct conjugates of SUMO, and we mutated SUMO conjugation sites in four proteins . This is the first attempt to immunoprecipitate a large fraction of the proteome of a eukaryote, and it demonstrates the utility of this method to identify post-translational modifications in the yeast proteome.

Biochemistry, 2004 Dec 21, 43(50), 15702 - 19
Expanding the functional repertoire of CTD kinase I and RNA polymerase II: novel phosphoCTD-associating proteins in the yeast proteome; Phatnani HP et al.; CTD kinase I (CTDK-I) of Saccharomyces cerevisiae is required for normal phosphorylation of the C-terminal repeat domain (CTD) on elongating RNA polymerase II . To elucidate cellular roles played by this kinase and the hyperphosphorylated CTD (phosphoCTD) it generates, we systematically searched yeast extracts for proteins that bound to the phosphoCTD made by CTDK-I in vitro . Initially, using a combination of far-western blotting and phosphoCTD affinity chromatography, we discovered a set of novel phosphoCTD-associating proteins (PCAPs) implicated in a variety of nuclear functions . We identified the phosphoCTD-interacting domains of a number of these PCAPs, and in several test cases (namely, Set2, Ssd1, and Hrr25) adduced evidence that phosphoCTD binding is functionally important in vivo . Employing surface plasmon resonance (BIACORE) analysis, we found that recombinant versions of these and other PCAPs bind preferentially to CTD repeat peptides carrying SerPO(4) residues at positions 2 and 5 of each seven amino acid repeat, consistent with the positional specificity of CTDK-I in vitro {Jones, J . C., et al . (2004) J . Biol . Chem . 279, 24957-24964} . Subsequently, we used a synthetic CTD peptide with three doubly phosphorylated repeats (2,5P) as an affinity matrix, greatly expanding our search for PCAPs . This resulted in identification of approximately 100 PCAPs and associated proteins representing a wide range of functions (e.g., transcription, RNA processing, chromatin structure, DNA metabolism, protein synthesis and turnover, RNA degradation, snRNA modification, and snoRNP biogenesis) . The varied nature of these PCAPs and associated proteins points to an unexpectedly diverse set of connections between Pol II elongation and other processes, conceptually expanding the role played by CTD phosphorylation in functional organization of the nucleus.

Nat Genet, 2005 Jan, 37(1), 77 - 83 Epub 2004 Dec 12.
Modular epistasis in yeast metabolism; Segre D et al.; Epistatic interactions, manifested in the effects of mutations on the phenotypes caused by other mutations, may help uncover the functional organization of complex biological networks . Here, we studied system-level epistatic interactions by computing growth phenotypes of all single and double knockouts of 890 metabolic genes in Saccharomyces cerevisiae, using the framework of flux balance analysis . A new scale for epistasis identified a distinctive trimodal distribution of these epistatic effects, allowing gene pairs to be classified as buffering, aggravating or noninteracting . We found that the ensuing epistatic interaction network could be organized hierarchically into function-enriched modules that interact with each other 'monochromatically' (i.e., with purely aggravating or purely buffering epistatic links) . This property extends the concept of epistasis from single genes to functional units and provides a new definition of biological modularity, which emphasizes interactions between, rather than within, functional modules . Our approach can be used to infer functional gene modules from purely phenotypic epistasis measurements.

J Biol Chem . 2004 Dec 8; {Epub ahead of print}
Expression of a recQ helicase homolog affects progression through crisis in fission yeast lacking telomerase; Mandell JG et al.; RecQ helicases play roles in telomere maintenance in cancerous human cells using the alternative lengthening of telomeres (ALT) mechanism and in budding yeast lacking telomerase . Fission yeast lacking the catalytic subunit of telomerase (trt1+) up-regulate the expression of a previously uncharacterized sub-telomeric open reading frame (ORF) as survivors emerge from crisis . Here we show that this ORF encodes a protein with homology to RecQ helicases such as the human Bloom's and Werner's syndrome proteins, and that copies of the helicase gene are present on multiple chromosome ends . Characterization of the helicase transcript revealed a 7.6 kb RNA that was associated with polyribosomes, suggesting it is translated . A 3.6 kb domain of the helicase gene predicted to encode the region with catalytic activity was cloned, and both native and mutant forms of this domain were overexpressed in trt1- cells as they progressed through crisis . Overexpression of the native form caused cells to recover from crisis earlier than cells with a vector-only control, whereas overexpression of the mutant form caused delayed recovery from crisis . Taken together, the sequence homology, functional analysis, and site-directed mutagenesis indicate that the protein is likely a second fission yeast RecQ helicase (in addition to Rqh1) that participates in telomere metabolism during crisis . These results strengthen the notion that in multiple organisms RecQ helicases contribute to survival after telomere damage.

Eukaryot Cell, 2004 Dec, 3(6), 1664 - 73
Prm1 prevents contact-dependent lysis of yeast mating pairs; Jin H et al.; Membrane fusion requires localized destabilization of two phospholipid bilayers, but unrestrained membrane destabilization could result in lysis . prm1 mutant yeast cells have a defect at the plasma membrane fusion stage of mating that typically results in the accumulation of prezygotes that have fingers of membrane-bound cytoplasm projecting from one cell of each pair into its mating partner in the direction of the osmotic gradient between the cells . However, some prm1 mating pairs fuse successfully whereas the two cells in other prm1 mating pairs simultaneously lyse . Lysis only occurs if both mating partners are prm1 mutants . Osmotic stabilization does not protect prm1 mating pairs from lysis, indicating that lysis is not caused by a cell wall defect . prm1 mating pairs without functional mitochondria still lyse, ruling out programmed cell death . No excess lysis was found after pheromone treatment of haploid prm1 cells, and lysis did not occur in mating pairs when prm1 was combined with the fus1 and fus2 mutations to block cell wall remodeling . Furthermore, short (<1 microm) cytoplasmic microfingers indicating the completion of cell wall remodeling appeared immediately before lysis . In combination, these results demonstrate that plasma membrane contact is a prerequisite for lysis . Cytoplasmic microfingers are unlikely to cause lysis since most prm1 mating pairs with microfingers do not lyse, and microfingers were also detected before fusion in some wild-type mating pairs . The lysis of prm1 mutant mating pairs suggests that the Prm1 protein stabilizes the membrane fusion event of yeast mating.

DNA Repair (Amst), 2005 Feb 3, 4(2), 253 - 61
The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis; Sia EA et al.; Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae . With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously . Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5) . Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination . However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined . We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability . These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.

Biosens Bioelectron, 2005 Jan 15, 20(7), 1285 - 90
A novel l-lactate-selective biosensor based on flavocytochrome b(2) from methylotrophic yeast Hansenula polymorpha; Smutok O et al.; A novel amperometric biosensor highly selective to l-lactate has been developed using l-lactate-cytochrome c oxidoreductase (flavocytochrome b(2)) isolated for the first time from thermotolerant methylotrophic yeast Hansenula polymorpha as biorecognition element . Different immobilization methods and low-molecular free-diffusing redox mediators have been tested for optimising the electrochemical communication between the immobilized enzyme and the electrode surface . Moreover, the possibility of direct electron transfer from the reduced form of FCb(2) to carbon electrodes has been evaluated . The bioanalytical properties of FCb(2)-based biosensors, such as signal rise time, dynamic range, dependence of the sensor output on the pH value, the temperature and the storage stability were investigated, and the proposed biosensor demonstrated a very fast response and a high sensitivity and selectivity for l-lactate determination.

Methods, 2005 Jan, 35(1), 54 - 63
Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z; Fukada M et al.; Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood . This is due to a lack of information concerning the physiological substrates of the respective PTPs . Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro . The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins . Therefore, the development of a standard method applicable to all PTPs has long been awaited . We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait . This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs . Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions . The identification of physiological substrates will shed light on the physiological functions of individual PTPs.

J Mol Biol, 2005 Jan 28, 345(4), 855 - 67
Many faces of the unfolded state: conformational heterogeneity in denatured yeast cytochrome C; Pletneva EV et al.; We have measured fluorescence energy-transfer (FET) kinetics from a dansyl fluorophore (Dns) introduced by derivatization of a Cys side-chain to the Fe(III) heme covalently attached to unfolded yeast iso-1 cytochrome c (cyt) . To gain a global picture of the unfolded state, we examined variants with the fluorophore attached on three different helices (K4C, E66C, K99C) and in three different loops (H39C, D50C, L85C) . Analysis of the FET kinetics data gave distributions of distances between the fluorescent donor and acceptor; these distributions demonstrate that the guanidine hydrochloride (GuHCl)-denatured polypeptide ensemble is not a simple random coil . Although misligation imposes some constraints, it is not the only source of structural complexity in the unfolded protein . Our FET kinetics data reveal a high degree of heterogeneity in the unfolded ensemble of cytochrome c . We detect relatively large populations of compact structures in unfolded Dns(C50)cyt, Dns(C39)cyt, and Dns(C66)cyt . These structures likely play a role in forming a hydrophobic core during the folding process.

EMBO J . 2004 Dec 8;23(24):4876.
Mutations in yeast Rad51 that partially bypass the requirement for Rad55 and Rad57 in DNA repair by increasing the stability of Rad51-DNA complexes; Fortin GS et al.; Correction to: The EMBO Journal (2002) 21, 3160-3170 . doi:10.1093/emboj/cdf293.

Sci STKE . 2004 Dec 7;2004(262):tr12.
Phosphorelay signaling in yeast in response to changes in osmolarity; Santos JL et al.; In the yeast, Saccharomyces cerevesiae, phosphorelay signaling systems that involve a three-step His-Asp-His-Asp phosphotransfer are involved in transmitting signals in response to cellular stress . The animation shows one example of such a phosphorelay system involved in yeast responses to changes in osmolarity . Under conditions of low osmolarity, a histidine-aspartate phosphorelay pathway transmits information that deactivates one signaling pathway and activates gene expression through another pathway . In response to high osmolarity, the Sln1 kinase that initiates the phosphorelay is inhibited and the Hog1 mitogen-activated protein kinase cascade is active.

Yeast, 2005 Jan 15, 22(1), 43 - 56
Biochemical and genetic characterization of Yra1p in budding yeast; Kashyap AK et al.; Yra1p and its vertebrate homologues bind to the mRNA export factor Mex67p/TAP and are thought to play a role in mRNA export in vivo . To further characterize Yra1p, we used immunoaffinity chromatography to purify endogenous Yra1p complexes . These experiments demonstrated that two importin beta homologues (Kap123p and Pse1p) and the poly A tail-binding proteins Pab1p and Nab2p associate with Yra1p . The other major proteins that associate with Yra1p include proteins involved in mRNA and rRNA processing and the Yra1p-related protein Yra2p . Additional biochemical and genetic experiments suggest a close functional relationship between Yra1p and Yra2p . We generated a temperature-sensitive allele of YRA1 and used it to demonstrate that cells which lack the function of both Yra1p and Yra2p are able to exit a G(0) arrest and go through several rounds of cell division before arresting . We also identified high-copy suppressors of the yra1-2 temperature-sensitive growth defect . These include SUB2, a splicing factor important in mRNA export, ULP1, a nuclear cysteine protease localized to the nuclear pore and involved in Smt3p/SUMO processing, and YRA2 . Taken together, these results suggest that Yra1p has roles in diverse RNA processing events in addition to a role in mRNA export . Copyright (c) 2004 John Wiley & Sons, Ltd.

Int J Mol Med, 2005 Jan, 15(1), 123 - 7
Computationally analyzing the possible biological function of YJL103C--an ORF potentially involved in the regulation of energy process in yeast; Deng Y et al.; Although the complete genomes of a number of organisms have been sequenced, the biological functions of many genes are still not known . Because experimentally studying the functions of those genes one by one requires tremendous time, it is vital to use published resources like microarray gene expression data for computational analysis of gene functions . One example is YJL103C, a yeast gene of unknown function in the Saccharomyces Genome Database (SGD) . It is possible to quickly infer its biological function by computational analysis . In this study, we present an efficient model to explore the biological function of a novel gene using microarray data . We showed that the expression pattern of YJL103C is most similar to the genes in the energy group and respiratory chain subgroup . We further found that YJL103C contains a HAP2,3,4 box in its promoter region and a cytochrome C heme-binding signature in its protein sequence . Our findings define a potential role for YJL103C in the regulation of energy metabolism, specifically in the process of oxidative phosphorylation . Similar bioinformatics methods can be applied to infer the biological functions of other novel genes in organisms for which microarray data are available . In this work, we selected a single gene of unknown function as a case study . By focusing on the power of computer analysis and bioinformatics on the available microarray data, we have determined the likely biological function of YJL103C . Our study provides a method by which to explore the potential function of other genes currently annotated as having an unknown function in any organism for which global gene expression data are available.

Microbiology, 2004 Dec, 150(Pt 12), 4157 - 70
PST1 and ECM33 encode two yeast cell surface GPI proteins important for cell wall integrity; Pardo M et al.; Pst1p was previously identified as a protein secreted by yeast regenerating protoplasts, which suggests a role in cell wall construction . ECM33 encodes a protein homologous to Pst1p, and both of them display typical features of GPI-anchored proteins and a characteristic receptor L-domain . Pst1p and Ecm33p are both localized to the cell surface, Pst1p being at the cell membrane and possibly also in the periplasmic space . Here, the characterization of pst1Delta, ecm33Delta and pst1Delta ecm33Delta mutants is described . Deletion of ECM33 leads to a weakened cell wall, and this defect is further aggravated by simultaneous deletion of PST1 . As a result, the ecm33Delta mutant displays increased levels of activated Slt2p, the MAP kinase of the cell integrity pathway, and relies on a functional Slt2-mediated cell integrity pathway to ensure viability . Analyses of model glycosylated proteins show glycosylation defects in the ecm33Delta mutant . Ecm33p is also important for proper cell wall ultrastructure organization and, furthermore, for the correct assembly of the mannoprotein outer layer of the cell wall . Pst1p seems to act in the compensatory mechanism activated upon cell wall damage and, in these conditions, may partially substitute for Ecm33p.

J Electron Microsc (Tokyo), 2004, 53(5), 563 - 566
Application of a FIB-STEM system for 3D observation of a resin-embedded yeast cell; Kamino T et al.; The combination of a focused ion beam (FIB) system and a scanning transmission electron microscope (STEM) has been applied to the three-dimensional (3D) observation of a resin-embedded yeast cell . Using a FIB microsampling technique, a sample with a thickness of tens of micrometres was extracted from a resin-embedded block sample . The extracted sample was transferred to a FIB-STEM-compatible specimen rotation holder and trimmed by FIB milling for 3D STEM observation . Although the FIB milling was carried out at an operating voltage of 40 KV, the sample was cross sectioned without forming a harmful damage layer on its surface . Cell structures, such as cell wall, cell membrane, mitochondria, peroxisomes, endoplasmic reticulum and vacuoles, were observed clearly in a pillar-shaped sample of 20 microm long, 4 microm wide and 3 microm deep.

FEBS Lett, 2004 Dec 3, 578(1-2), 58 - 62
The Ppz protein phosphatases regulate Trk-independent potassium influx in yeast; Ruiz A et al.; The Ppz protein phosphatases have been recently shown to negatively regulate the major potassium transport system in the yeast Saccharomyces cerevisiae, encoded by the TRK1 and TRK2 genes . We have found that, in the absence of the Trk system, Ppz mutants require abnormally high concentrations of potassium to proliferate . This can be explained by the observation that trk1 trk2 ppz1 or trk1 trk2 ppz1 ppz2 strains display a very poor rubidium uptake, with markedly increased Km values . These cells are very sensitive to the presence of several toxic cations in the medium, such as hygromicyn B or spermine, but not to lithium or sodium cations . At limiting potassium concentrations, addition of EGTA to the medium improves growth of these mutants . Therefore, our results indicate that, in addition to their role in regulating Trk potassium transporters, Ppz phosphatases (essentially Ppz1), positively affect the residual low affinity potassium transport mechanisms in yeast . These findings may provide a new way to elucidate the molecular nature of the low affinity potassium uptake system in yeast as well as a useful model to analyze the function of plant or mammalian potassium channels through heterologous expression in yeast.

Arch Biochem Biophys, 2005 Jan 1, 433(1), 335 - 40
Effects of pressure on deuterium isotope effects of yeast alcohol dehydrogenase using alternative substrates; Park H et al.; Hydrostatic pressure causes biphasic effects on the oxidation of alcohols by yeast alcohol dehydrogenase as expressed on the kinetic parameter V/K which measures substrate capture . Moderate pressure increases capture by activating hydride transfer, whose transition-state must therefore have a smaller volume than the free alcohol plus the capturing form of enzyme, with DeltaV(double dagger)=-30 mL mol(-1) for isopropanol . A comparison of these effects with those on the oxidation of deutero-isopropanol generates a monophasic decrease in the intrinsic isotope effect; therefore, the volume of activation for the transition-state of deuteride transfer must be even more negative, by 7.6 mL mol(-1) . The pressure data extrapolate and factor the kinetic isotope effect into a semi-classical reactant-state component, with a null value of k(H)/k(D)=1, and a transition-state component of Q(H)/Q(D)=4, suggestive of hydrogen tunneling . Pressures above 1.5 kbar decrease capture by favoring a minor conformation of enzyme which binds nicotinamide adenine dinucleotide (NAD(+)) less tightly . This inactive conformation has a smaller volume than active E-NAD(+), with a difference of 74 mL mol(-1) and an equilibrium constant of 93 between them, at one atmosphere of pressure . These results are virtually identical to those obtained with benzyl alcohol and give credence to this method of analysis . Moreover, qualitatively similar results with greater pressure sensitivity but less precision are obtained using ethanol as a substrate, only with pressure driving the value of the isotope effect to a value less than (D)k=1.03 directly, without extrapolation . The ethanol data verify the most surprising finding of these studies, namely that the entire kinetic isotope effect arises from a transition-state phenomenon.

Genetics, 2004 Nov, 168(3), 1763 - 71
Uncovering novel cell cycle players through the inactivation of securin in budding yeast; Sarin S et al.; Budding yeast securin/Pds1p, an inhibitor of the anaphase activator separase/Esp1p, is involved in several checkpoint pathways and in promoting Esp1p's nuclear localization . Using a modified synthetic genetic array (SGA) screen for genes that become essential in the absence of Pds1p, we uncovered roles for uncharacterized genes in cell cycle processes, including Esp1p activation.

Genetics, 2004 Nov, 168(3), 1219 - 30
The budding yeast mei5 and sae3 proteins act together with dmc1 during meiotic recombination; Tsubouchi H et al.; Here we provide evidence that the Mei5 and Sae3 proteins of budding yeast act together with Dmc1, a meiosis-specific, RecA-like recombinase . The mei5 and sae3 mutations reduce sporulation, spore viability, and crossing over to the same extent as dmc1 . In all three mutants, these defects are largely suppressed by overproduction of Rad51 . In addition, mei5 and sae3, like dmc1, suppress the cell-cycle arrest phenotype of the hop2 mutant . The Mei5, Sae3, and Dmc1 proteins colocalize to foci on meiotic chromosomes, and their localization is mutually dependent . The localization of Rad51 to chromosomes is not affected in either mei5 or sae3 . Taken together, these observations suggest that the Mei5 and Sae3 proteins are accessory factors specific to Dmc1 . We speculate that Mei5 and Sae3 are necessary for efficient formation of Dmc1-containing nucleoprotein filaments in vivo.

Genetics, 2004 Nov, 168(3), 1189 - 203
Compartmentalization of the yeast meiotic nucleus revealed by analysis of ectopic recombination; Schlecht HB et al.; As yeast cells enter meiosis, chromosomes move from a centromere-clustered (Rabl) to a telomere-clustered (bouquet) configuration and then to states of progressive homolog pairing where telomeres are more dispersed . It is uncertain at which stage of this process sequences commit to recombine with each other . Previous analyses using recombination between dispersed homologous sequences (ectopic recombination) support the view that, on average, homologs are aligned end to end by the time of commitment to recombination . We have undertaken further analyses incorporating new inserts, chromosome rearrangements, an alternate mode of recombination initiation, and mutants that disrupt nuclear structure or telomere metabolism . Our findings support previous conclusions and reveal that distance from the nearest telomere is an important parameter influencing recombination between dispersed sequences . In general, the farther dispersed sequences are from their nearest telomere, the less likely they are to engage in ectopic recombination . Neither the mode of initiating recombination nor the formation of the bouquet appears to affect this relationship . We suggest that aspects of telomere localization and behavior influence the organization and mobility of chromosomes along their entire length, during a critical period of meiosis I prophase that encompasses the homology search.

BMC Mol Biol . 2004 Dec 03;5(1):21.
Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase delta subunit Cdc27; Gray FC et al.; BACKGROUND: DNA polymerases alpha and delta play essential roles in the replication of chromosomal DNA in eukaryotic cells . DNA polymerase alpha (Pol alpha)-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase delta (Pol delta) is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase epsilon (Pol epsilon) . RESULTS: Here it is shown that the catalytic subunit of Pol alpha, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol delta, both in vivo and in vitro . Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA . Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM), in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals . Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function . CONCLUSIONS: A short protein sequence motif (DPIM) has been identified as mediating Pol alpha-Pol delta interactions in fission yeast . Despite being conserved across species, mutational analysis indicates the DPIM does not play an essential role in vivo, suggesting that interaction between the two polymerases is also non-essential.

Nucleic Acids Res, 2004, 32(21), 6367 - 77 Print 2004.
Genetics of lagging strand DNA synthesis and maturation in fission yeast: suppression analysis links the Dna2-Cdc24 complex to DNA polymerase delta; Tanaka H et al.; The Cdc24 protein is essential for the completion of chromosomal DNA replication in fission yeast . Although its precise role in this process is unclear, Cdc24 forms a complex with Dna2, a conserved endonuclease-helicase implicated in the removal of the RNA-DNA primer during Okazaki fragment processing . To gain further insights into Cdc24-Dna2 function, we screened for chromosomal suppressors of the temperature-sensitive cdc24-M38 allele and mapped the suppressing mutations into six complementation groups . Two of these mutations defined genes encoding the Pol3 and Cdc27 subunits of DNA polymerase delta . Sequence analysis revealed that all the suppressing mutations in Cdc27 resulted in truncation of the protein and loss of sequences that included the conserved C-terminal PCNA binding motif, previously shown to play an important role in maximizing enzyme processivity in vitro . Deletion of this motif is shown to be sufficient for suppression of both cdc24-M38 and dna2-C2, a temperature-sensitive allele of dna2(+), suggesting that disruption of the interaction between Cdc27 and PCNA renders the activity of the Cdc24-Dna2 complex dispensable.

Biochim Biophys Acta, 2004 Dec 6, 1659(2-3), 197 - 205
Application of the yeast Yarrowia lipolytica as a model to analyse human pathogenic mutations in mitochondrial complex I (NADH:ubiquinone oxidoreductase); Kerscher S et al.; While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity . Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I . For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria . Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation . In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated K(M) values, and/or elevated I(50) values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level . Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core . This supports the proposed structural model and at the same time identifies novel domains critical for catalysis . Thus, Y . lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.

Biochim Biophys Acta, 2004 Dec 6, 1659(2-3), 153 - 9
Defects in the biosynthesis of mitochondrial heme c and heme a in yeast and mammals; Moraes CT et al.; Defects in heme biosynthesis have been associated with a large number of diseases, but mostly recognized in porphyrias, which are neurovisceral or cutaneous disorders caused by the accumulation of biosynthetic intermediates . However, defects in the maturation of heme groups that are part of the oxidative phosphorylation system are now also recognized as important causes of disease . The electron transport chain contains heme groups of the types a, b and c, all of which are directly involved in electron transfer reactions . In this article, we review the effect of mutations in enzymes involved in the maturation of heme a (the prosthetic group of cytochrome c oxidase) and heme c (the prosthetic group of cytochrome c) both in yeast and in humans . COX10 and COX15 are two genes, initially identified in Saccharomyces cerevisiae that have been found to cause infantile cytochrome c oxidase deficiency in humans . They participate in the farnesylation and hydroxylation of heme b, steps that are necessary for the formation of heme a, the prosthetic group required for cytochrome oxidase assembly and activity . Deletion of the cytochrome c heme lyase gene in a single allele has also been associated with a human disease, known as Microphthalmia with Linear Skin defects (MLS) syndrome . The cytochrome c heme lyase is necessary to covalently attach the heme group to the apocytochrome c polypeptide . The production of mouse models recapitulating these diseases is providing novel information on the pathogenesis of clinical syndromes.

BMC Pharmacol . 2004 Dec 2;4(1):31 {Epub ahead of print}
Characterisation of DNA damage and cytotoxicity induced by the bisdioxopiperazine anti-cancer agent ICRF-187 (dexrazoxane) in yeast and mammalian cells; Jensen LH et al.; BACKGROUND: Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA . It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells . In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches . In addition, the well-established topoisomerase II poison m-AMSA is used for comparison . RESULTS: By utilizing a panel of Saccharomyces cerevisiae deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187 . However, sensitivity towards m-AMSA depended much more on this pathway . In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA . Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more . Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs . DNA-PKcs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs . While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced gamma-H2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA . Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187 . Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells . CONCLUSION: Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms.

J Biol Chem . 2004 Dec 1; {Epub ahead of print}
Kinetic parameters and metabolic contributions of yeast mitochondrial and cytosolic NADP+-specific isocitrate dehydrogenase; Contreras-Shannon V et al.; To compare kinetic properties of homologous isozymes of NADP(+)-specific isocitrate dehydrogenase, histidine-tagged forms of yeast mitochondrial (IDP1) and cytosolic (IDP2) enzymes were expressed and purified . The isozymes were found to share similar apparent affinities for cofactor . However, with respect to isocitrate, IDP1 had an apparent K(m) value ~seven-fold lower than that of IDP2 while, with respect to alpha-ketoglutarate, IDP2 had an apparent K(m) value ~ten-fold lower than that of IDP1 . Similar K(m) values for substrates and for cofactors in decarboxylation and carboxylation reactions were obtained for IDP2, suggesting a capacity for bidirectional catalysis in vivo . Concentrations of isocitrate and alpha-ketoglutarate measured in extracts from the parental strain were found to be similar with growth on different carbon sources . For mutant strains lacking IDP1, IDP2, and/or the mitochondrial NAD(+)-specific isocitrate dehydrogenase (IDH), metabolite measurements indicated that major cellular flux is through the IDH-catalyzed reaction in glucose-grown cells and through the IDP2-catalyzed reaction in cells grown with a nonfermentable carbon source (glycerol and lactate) . A substantial cellular pool of alpha-ketoglutarate is attributed to IDH function during glucose growth, and to both IDP1 and IDH function during growth on glycerol/lactate . Complementation experiments using a strain lacking IDH demonstrated that overexpression of IDP1 partially compensated for the glutamate auxotrophy associated with loss of IDH . Collectively, these results suggest an ancillary role for IDP1 in cellular glutamate synthesis and a role for IDP2 in equilibrating and maintaining cellular levels of isocitrate and alpha-ketoglutarate.

Mol Cell Biol, 2004 Dec, 24(24), 10965 - 74
Evidence for histone eviction in trans upon induction of the yeast PHO5 promoter; Korber P et al.; The yeast PHO5 promoter is a model system for the role of chromatin in eukaryotic gene regulation . Four positioned nucleosomes in the repressed state give way to an extended DNase I hypersensitive site upon induction . Recently this hypersensitive site was shown to be devoid of histone DNA contacts . This raises the mechanistic question of how histones are removed from the promoter . A displacement in trans or movement in cis, the latter according to the well established nucleosome sliding mechanism, are the major alternatives . In this study, we embedded the PHO5 promoter into the context of a small plasmid which severely restricts the space for nucleosome sliding along the DNA in cis . Such a construct would either preclude the chromatin transition upon induction altogether, were it to occur in cis, or gross changes in chromatin around the plasmid would be the consequence . We observed neither . Instead, promoter opening on the plasmid was indistinguishable from opening at the native chromosomal locus . This makes a sliding mechanism for the chromatin transition at the PHO5 promoter highly unlikely and points to histone eviction in trans.

J Cell Sci, 2004 Dec 15, 117(Pt 26), 6447 - 54 Epub 2004 Nov 30.
Sem1, the yeast ortholog of a human BRCA2-binding protein, is a component of the proteasome regulatory particle that enhances proteasome stability; Funakoshi M et al.; Degradation of polyubiquitinated proteins by the proteasome often requires accessory factors; these include receptor proteins that bind both polyubiquitin chains and the regulatory particle of the proteasome . Overproduction of one such factor, Dsk2, is lethal in Saccharomyces cerevisiae and we show here that this lethality can be suppressed by mutations in SEM1, a gene previously recognized as an ortholog of the human gene encoding DSS1, which binds the BRCA2 DNA repair protein . Yeast sem1 mutants accumulate polyubiquitinated proteins, are defective for proteasome-mediated degradation and cannot grow under various stress conditions . Moreover, sem1 is synthetically lethal with mutations in proteasome subunits . We show that Sem1 is a component of the regulatory particle of the proteasome, specifically the lid subcomplex . Loss of Sem1 impairs the stability of the 26S proteasome and sem1Delta defects are greatly enhanced by simultaneous deletion of RPN10 . The Rpn10 proteasome subunit appears to function with Sem1 in maintaining the association of the lid and base subcomplexes of the regulatory particle . Our data suggest a potential mechanism for this protein-protein stabilization and also suggest that an intact proteasomal regulatory particle is required for responses to DNA damage.

J Theor Biol, 2005 Feb 7, 232(3), 385 - 398
Yeast glycolytic oscillations that are not controlled by a single oscillophore: a new definition of oscillophore strength; Reijenga KA et al.; Biochemical oscillations, such as glycolytic oscillations, are often believed to be caused by a single so-called 'oscillophore' . The main characteristics of yeast glycolytic oscillations, such as frequency and amplitude, are however controlled by several enzymes . In this paper, we develop a method to quantify to which extent any enzyme determines the occurrence of oscillations . Principles extrapolated from metabolic control analysis are applied to calculate the control exerted by individual enzymes on the real and imaginary parts of the eigenvalues of the Jacobian matrix . We propose that the control exerted by an enzyme on the real part of the smallest eigenvalue, in terms of absolute value, quantifies to which extent that enzyme contributes to the emergence of instability . Likewise the control exerted by an enzyme on the imaginary part of complex eigenvalues may serve to quantify the extent to which that enzyme contributes to the tendency of the system to oscillate . The method was applied both to a core model and to a realistic model of yeast glycolytic oscillations . Both the control over stability and the control over oscillatory tendency were distributed among several enzymes, of which glucose transport, pyruvate decarboxylase and ATP utilization were the most important . The distributions of control were different for stability and oscillatory tendency, showing that control of instability does not imply control of oscillatory tendency nor vice versa . The control coefficients summed up to 1, suggesting the existence of a new summation theorem . These results constitute proof that glycolytic oscillations in yeast are not caused by a single oscillophore and provide a new, subtle, definition for the oscillophore strength of an enzyme.

Biochim Biophys Acta, 2004 Nov 29, 1695(1-3), 89 - 111
Ubiquitin and endocytic internalization in yeast and animal cells; Dupre S et al.; Endocytosis is involved in a wide variety of cellular processes, and the internalization step of endocytosis has been extensively studied in both lower and higher eukaryotic cells . Studies in mammalian cells have described several endocytic pathways, with the main emphasis on clathrin-dependent endocytosis . Genetic studies in yeast have underlined the critical role of actin and actin-binding proteins, lipid modification, and the ubiquitin conjugation system . The combined results of studies of endocytosis in higher and lower eukaryotic cells reveal an interesting interplay in the two systems, including a crucial role for ubiquitin-associated events . The ubiquitylation of yeast cell-surface proteins clearly acts as a signal triggering their internalization . Mammalian cells display variations on the common theme of ubiquitin-linked endocytosis, according to the cell-surface protein considered . Many plasma membrane channels, transporters and receptors undergo cell-surface ubiquitylation, required for the internalization or later endocytic steps of some cell-surface proteins, whereas for others, internalization involves interaction with the ubiquitin conjugation system or with ancillary proteins, which are themselves ubiquitylated . Epsins and Eps15 (or Eps15 homologs), are commonly involved in the process of endocytosis in all eukaryotes, their critical role in this process stemming from their capacity to bind ubiquitin, and to undergo ubiquitylation.

J Colloid Interface Sci, 2005 Jan 15, 281(2), 261 - 6
Biosorption of chromium(VI) and arsenic(V) onto methylated yeast biomass; Seki H et al.; Yeast biomass was methylated in a 0.1 M HCl methyl alcohol solution at room temperature and the methylated yeast (MeYE) was applied to the adsorptive separation of Cr(VI) and As(V) anions from aqueous solutions . At near-neutral pH, while Cr(VI) and As(V) anions were scarcely adsorbed onto unmethylated yeast biomass, the amounts adsorbed increased with increasing methylation degree . The amount of Cr(VI) adsorbed onto MeYE was almost constant at pH 4-6 and decreased with increasing pH above pH 6 . The amount of As(V) adsorbed onto MeYE was rather lower than that of Cr(VI) and it had a peak at about pH 7 . A metal-binding model was used to describe the adsorption characteristics of Cr(VI) and As(V) on MeYE . The results showed that MeYE has two different types of adsorption sites . The saturated amount of Cr(VI) and As(V) adsorbed onto MeYE having methylation degree 0.94 was 0.55 mmol g(-1).

Traffic, 2005 Jan, 6(1), 56 - 65
Starvation Triggers the Delivery of the Endoplasmic Reticulum to the Vacuole via Autophagy in Yeast; Hamasaki M et al.; Autophagy is a survival mechanism necessary for eukaryotic cells to overcome nutritionally challenged environments . When autophagy is triggered, cells degrade nonselectively engulfed cytosolic proteins and free ribosomes that are evenly distributed throughout the cytoplasm . The resulting pool of free amino acids is used to sustain processes crucial for survival . Here we characterize an autophagic degradation of the endoplasmic reticulum (ER) under starvation conditions in addition to cytosolic protein degradation . Golgi membrane protein was not engulfed by the autophagosome under the same conditions, indicating that the uptake of ER by autophagosome was the specific event . Although the ER exists in a network structure that is mutually connected and resides predominantly around the nucleus and beneath the plasma membrane, most of autophagosome engulfed ER . The extent of the ER uptake by autophagy was nearly identical to that of the soluble cytosolic proteins . This phenomenon was explained by the appearance of fragmented ER membrane structures in almost all autophagosomes . Furthermore, ER dynamism is required for this process: ER uptake by autophagosomes occurs in an actin-dependent manner.

Genes Cells, 2004 Dec, 9(12), 1275 - 86
The Sec14 family glycerophospholipid-transfer protein is required for structural integrity of the spindle pole body during meiosis in fission yeast; Nakase Y et al.; The fission yeast spo20+ gene encodes a glycerophospholipid-transfer protein . spo20 mutants are unable to assemble the forespore membrane properly . Here we studied the structural integrity of the spindle pole body (SPB) in spo20-H6 mutants during meiosis . Meiotic cells expressing a GFP-tagged SPB marker protein, Spo15-GFP, showed an excess number of SPBs, some of which were not localized to the spindle poles and were termed 'pseudo-SPBs' . Formation of spindles for meiosis I was significantly delayed in spo20-H6 cells, although the morphology of spindles and segregation of the sister chromatids seemed normal . The SPB of spo20-H6 contained meiosis-specific outer plaques, though outermost layers were less evident . Time-lapse studies of spo20-H6 cells showed that the pseudo-SPBs originated from normal SPBs at the spindle poles during meiosis I . Among the SPB components tested, Spo15, Spo13, Sad1 and Cut12 were localized to the pseudo-SPBs, but Sid4 was not always present . Alp4, a component of the gamma-tubulin complex, was also present in about 40% of the pseudo-SPBs . The forespore membranes initiated from both the SPBs and the pseudo-SPBs . We conclude that Spo20 plays a role in maintaining the structural integrity of the meiotic SPB, besides supplying membrane vesicles for forespore membrane assembly.

Annu Rev Genet, 2004, 38, 233 - 71
Recombination proteins in yeast; Krogh BO et al.; The process of homologous recombination promotes error-free repair of double-strand breaks and is essential for meiosis . Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing radiation-induced DNA damage in Saccharomyces cerevisiae . The Rad52 group proteins are highly conserved among eukaryotes . Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity . Herein, we review recent genetic, biochemical, and structural analyses of the genes and proteins involved in recombination.

Biochemistry, 2004 Dec 7, 43(48), 15230 - 45
Proton donor in yeast pyruvate kinase: chemical and kinetic properties of the active site Thr 298 to Cys mutant; Susan-Resiga D et al.; The active site T298 residue of yeast pyruvate kinase (YPK), located in a position to serve potentially as the proton donor, was mutated to cysteine . T298C YPK was isolated and purified, and its enzymatic properties were characterized . Fluorescence and CD spectra indicate minor structural perturbations . A kinetic analysis of the Mg(2+)-activated enzyme demonstrates no catalytic activity in the absence of the heterotropic activator fructose 1,6-bisphosphate (FBP) . In the presence of Mg(2+) and FBP, T298C has approximately 20% of the activity of wild-type (wt) YPK . The activator constant for FBP increases by 1 order of magnitude compared to this constant with the wt enzyme . T298C shows positive cooperativity by FBP with a Hill coefficient of 2.6 (wt, n(H,FBP) = 1) . Mn(2+)-activated T298C behaves like Mn(2+)-activated wt YPK with a V(max) that is 20% of that for the wt enzyme with or without FBP . A pH-rate profile of T298C relative to that for wt YPK shows that pK(a,2) has shifted from 6.4 in wt to 5.5, indicating that the thiol group elicits an acidic pK shift . Inactivation of both wt and T298C by iodoacetate elicits a pseudo-first-order loss of activity with T298C being inactivated from 8 to 100 times faster than wt YPK . A pH dependence of the inactivation rate constant for T298C gives a value of 8.2, consistent with the pK for a thiol . Changes in fluorescence indicate that the T298C-Mg(2+) complex binds PEP, ADP, and both ligands together . This demonstrates that the lack of activity is not due to the loss of substrate binding but to the lack of ability to induce the proper conformational change . The mutation also induces changes in binding of FBP to all the relevant complexes . Binding of the metal and binding of PEP to the enzyme complexes are also differentially altered . Solvent isotope effects are observed for both wt and T298C . Proton inventory studies indicate that k(cat) is affected by a proton from water in the transition state and the effects are metal ion-dependent . The results are consistent with water being the active site proton donor . Active site residue T298 is not critical for activity but plays a role in the activation of the water and affects the pK that modulates catalytic activity.

Math Med Biol, 2004 Dec, 21(4), 369 - 95
Estimating the number of prions in yeast cells; Cole DJ et al.; Certain yeast cells contain proteins that behave like the mammalian prion PrP and are called yeast prions . The yeast prion protein Sup35p can exist in one of two stable forms, giving rise to phenotypes {PSI(+)} and {psi(-)} . If the chemical guanidine hydrochloride (GdnHCl) is added to a culture of growing {PSI(+)} cells, the proportion of {PSI(+)} cells decreases over time . This process is called curing and is due to a failure to propagate the prion form of Sup35p . We describe how curing can be modelled, and improve upon previous models for the underlying processes of cell division and prion segregation; the new model allows for asymmetric cell division and unequal prion segregation . We conclude by outlining plans for future experimentation and modelling.

Science, 2004 Nov 26, 306(5701), 1508 - 9
Pheromone signaling mechanisms in yeast: a prototypical sex machine; Wang Y et al.; The actions of many extracellular stimuli are elicited by complexes of cell surface receptors, heterotrimeric guanine nucleotide-binding proteins (G proteins), and mitogen-activated protein (MAP) kinase complexes . Analysis of haploid yeast cells and their response to peptide mating pheromones has produced important advances in our understanding of G protein and MAP kinase signaling mechanisms . Many of the components, their interrelationships, and their regulators were first identified in yeast . Current analysis of the pheromone response pathway (see the Connections Maps at Science's Signal Transduction Knowledge Environment) will benefit from new and powerful genomic, proteomic, and computational approaches that will likely reveal additional general principles that are applicable to more complex organisms.

J Chemother, 2004 Oct, 16(5), 468 - 73
Antifungal activity of amphotericin B and itraconazole against filamentous fungi: comparison of the Sensititre Yeast One and NCCLS M38--a reference methods; Carrillo-Munoz AJ et al.; The susceptibilities of 81 clinical isolates of Aspergillus spp., Fusarium spp., and Scedosporium spp., to amphotericin B and itraconazole were determined by the colorimetric microdilution method Sensititre and the reference microdilution method of NCCLS standard M38-A for filamentous fungi . No major discrepancies were found and agreement ranged between 86.4% to 84% and 69.1% to 86.4% for amphotericin B and itraconazole respectively at 48 h and 72 h of incubation by using the recommended endpoints . Within two two-fold dilutions, high levels of agreement were found in general for amphotericin B at 48 or 72 h (86.4 to 87.7%) and itraconazole (91.4 to 93.8%) . Relatively better agreement was found for itraconazole at 72 h of incubation and 48 for amphotericin B.

Nature, 2004 Nov 25, 432(7016), 517 - 22
The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II; Kim M et al.; The carboxy-terminal domain (CTD) of the RNA polymerase II (RNApII) largest subunit consists of multiple heptapeptide repeats with the consensus sequence YSPTSPS . Different CTD phosphorylation patterns act as recognition sites for the binding of various messenger RNA processing factors, thereby coupling transcription and mRNA processing . Polyadenylation factors are co-transcriptionally recruited by phosphorylation of CTD serine 2 (ref . 2) and these factors are also required for transcription termination . RNApII transcribes past the poly(A) site, the RNA is cleaved by the polyadenylation machinery, and the RNA downstream of the cleavage site is degraded . Here we show that Rtt103 and the Rat1/Rai1 5' --> 3' exonuclease are localized at 3' ends of protein coding genes . In rat1-1 or rai1Delta cells, RNA 3' to polyadenylation sites is greatly stabilized and termination defects are seen at many genes . These findings support a model in which poly(A) site cleavage and subsequent degradation of the 3'-downstream RNA by Rat1 trigger transcription termination.

Gene, 2004 Dec 8, 343, 181 - 90
Consecutive gene deletions in Mycobacterium smegmatis using the yeast FLP recombinase; Stephan J et al.; Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants, because there are only two efficient resistance markers available for allelic exchange experiments . We have established a system based on the Flp recombinase of the yeast Saccharomyces cerevisiae for use in the nonpathogenic model organism Mycobacterium smegmatis . This system consists of a hygromycin resistance cassette flanked by two Flp recognition targets (FRT) in direct orientation and a curable plasmid for expression of the flp gene . The FRT-hyg-FRT cassette was used on a suicide plasmid and on a conditionally replicating plasmid to delete two of the four known porin genes of M . smegmatis, mspA and mspC, respectively, by homologous recombination . The hyg gene was specifically removed from the chromosome of both mutants upon expression of the flp gene . Based on the marker-less mspC mutant strain, a double knock-out mutant lacking also mspA was obtained using the same strategy . Thus, by a fast and efficient two-step procedure, each of the porin genes was replaced by a single FRT site, which can be further used for site-specific integration . These results show that the Flp/FRT system is a suitable genetic tool for constructing unmarked mutations and for the analysis of redundant genes by consecutive gene deletions in M . smegmatis.

J Biol Chem . 2004 Nov 24; {Epub ahead of print}
Functional domains of the yeast chromatin protein SIN1p/SPT2p can bind four-way junction and crossing DNA structures; Novoseler M et al.; SIN1p/SPT2p is a yeast chromatin protein which, when mutated or deleted, alters the transcription of a family of genes, presumably by modulating local chromatin structure . In this study, we investigated the ability of different domains of this protein to bind four-way junction DNA (4WJDNA), since 4WJDNA can serve as a model for bent double helical DNA and for the crossed structure formed at the exit and entry of DNA to the nucleosomes . Sequence alignment of SIN1p/SPT2p homologues from eleven different yeast species showed conservation of several domains . We found that three domains of SIN1p/SPT2p fused to glutathione-S-transferase can each bind independently in a structure specific manner to 4WJDNA as measured in a gel mobility shift assay . A feature common to these domains is a cluster of positively charged amino acids . Modification of this cluster results in either abolishment of binding, or a change in the binding properties . One of the domains tested clearly bound superhelical DNA, though it failed to induce bending in a circulization assay . Poly-L-lysine, which may be viewed as a cluster of positively charged amino acids, bound 4WJDNA as well . Phenotypic analysis showed that disruption of any of these domains results in suppression of a his4-912d allele, indicating that each domain has functional significance . We propose that SIN1p/SPT2p is likely to modulate local chromatin structure by binding two strands of double stranded DNA at their crossover point.

Fish Shellfish Immunol, 2005 Apr, 18(4), 311 - 25
Short- and long-term effects of a dietary yeast beta-glucan (Macrogard) and alginic acid (Ergosan) preparation on immune response in sea bass (Dicentrarchus labrax); Bagni M et al.; The present study investigated the immunomodulatory activity of Ergosan, an algal extract containing alginic acid, and Macrogard, a yeast extract containing beta-glucans, on innate and specific immunity in sea bass (Dicentrarchus labrax) . Four cycles of experimental feeding using normal fish feed formulation (control group) supplemented with Ergosan (0.5%) or Macrogard (0.1%) were performed at 60-day intervals (15 days of treatment+45 days of suspension) . Serum complement, lysozyme, total proteins and heat shock protein (HSP) concentrations were measured at 15, 30 and 45 days from the end of the first 15-day feeding cycle (short term) and 45 days after the end of each feeding cycle over a 35-week period (long term) . The percentage of B- and T-lymphocytes in peripheral blood leucocytes and gut were measured over long-term trial . Significant elevation (P < 0.05) in serum complement activity occurred in sea bass fed with alginic acid and glucans, at 15 days from the end of first cycle of treatment . Significant elevation (P < 0.05) in serum lysozyme, gill and liver HSP concentration were observed in the same experimental groups at 30 days from the end of treatment, whereas a significant increase (P < 0.05) of complement activity was only observed in fish that received an Ergosan diet . At 45 days from the end of treatment, complement, lysozyme and HSP concentration did not differ among groups . Over the long-term period, no significant differences were observed in innate and specific immune parameters, survival, growth performances and conversion index in treated and control fish . A dramatic decrease of both innate and acquired immune parameters was observed during the winter season in all groups, followed by a partial recovery when water temperature increased . Reduction in complement and lysozyme activities was significatively correlated (p < 0.01) to water temperature variation . The results suggested the potential of alginic acid and beta-glucans to activate some innate immune responses in sea bass, and particularly under conditions of immunodepression related to environmental stress.

J Chromatogr A, 2004 Nov 5, 1055(1-2), 177 - 84
Comparison of extraction methods for quantitation of methionine and selenomethionine in yeast by species specific isotope dilution gas chromatography-mass spectrometry; Yang L et al.; Fourteen extraction methods commonly cited in the literature were evaluated for the quantitation of methionine (Met) and selenomethionine (SeMet) in a yeast candidate certified reference material (CRM) . Species specific isotope dilution (ID) gas chromatography-mass spectrometry (GC-MS) was utilized to effectively compensate for potential errors, such as losses during derivatization and clean up steps . Despite different extraction methods, the same derivatization procedure using methyl chloroformate was applied with a single exception, which was based on digestion with cyanogen bromide with 2% SnCl2 in 0.1 M HCl . Significant differences in measured Met and SeMet concentrations were obtained when different extraction methods were used . A 4 M methanesulfonic acid reflux digestion was found to be the most efficient for both analytes . Digestion with CNBr with 2% SnCl2 in 0.1 M HCl for the determination of SeMet showed the second highest extraction efficiency . Despite frequent use of enzymatic hydrolysis for the extraction of SeMet from yeast, very low extraction efficiencies for both analytes were obtained for four of eight tested methods . Among these, the highest extraction efficiencies for both analytes were obtained using 20mg pronase and 10mg lipase with incubation at 37 degrees C for 24 h . However, recoveries remained nearly 30 and 50% lower for Met and SeMet, respectively, compared to extraction with methanesulfonic acid . Lowest extraction efficiencies for both analytes were obtained when HCl or tetramethylammonium hydroxide (TMAH) digestions were used . Efficient extraction was also achieved using 200 mg (or 400 mg) of protease XIV with incubation at 37 degrees C for 72 h (or 24 h) . Concentrations of 3331+/-45 and 3334+/-39 microg g(-1) (mean and one standard deviation, n = 4) for SeMet were obtained using 200 mg (72 h incubation) and 400 mg (24 h incubation) of protease XIV, respectively, in agreement with a value of 3404+/-38 microg g(-1) obtained using a methanesulfonic acid reflux.

Biotechniques, 2004 Nov, 37(5), 840 - 3
"One plate/three-reporter" assay format for the detection and validation of yeast two-hybrid interactions; Evans DR et al.; We describe a novel assay format for the Gal4-based yeast two-hybrid-system, in which the readout from three different reporter genes is measured sequentially in a single microplate . Activation of the URA3, MEL1, and lacZ reporters in response to a protein-protein interaction is monitored by measuring sequentially: (i) growth in medium lacking uracil, (ii) alpha-galactosidase activity, and (iii) beta-galactosidase . The data thus generated permit elimination of many false positive signals and provide a preliminary measurement of reporter activation-strength that may be confirmed by further analysis . The assay procedure is inexpensive and requires few liquid-handling steps . It is appropriate for automated high-throughput interaction mating assays, validation of putative interactor strains and hybrid-protein self-activator tests.

Prostaglandins Other Lipid Mediat, 2004 Oct, 74(1-4), 139 - 46
Report on the discovery of a novel 3-hydroxy oxylipin cascade in the yeast Saccharomycopsis synnaedendra; Sebolai OM et al.; A novel cascade of 3-hydroxy fatty acids was discovered in the yeast Saccharomycopsis synnaedendra . The cascade, probably derived from incomplete beta-oxidation, comprises both even and uneven carbon numbered as well as saturated and unsaturated 3-hydroxy oxylipins . This yeast may now be used as model to further study the metabolism of these compounds as well as their biotechnological production.

Curr Genet, 2004 Dec, 46(6), 331 - 42 Epub 2004 Nov 19.
Differential regulation of Tec1 by Fus3 and Kss1 confers signaling specificity in yeast development; Bruckner S et al.; Transcriptional regulation by mitogen-activated protein (MAP) kinase signaling cascades is a major control mechanism for eukaryotic development . In budding yeast, Fus3 and Kss1 are two MAP kinases that control two distinct developmental programs-mating and invasive growth . We investigated whether signal-specific activation of mating and invasive growth involves regulation of the transcription factor Tec1 by Fus3 and Kss1 . We present evidence that, during mating, Fus3 phosphorylates Tec1 to downregulate this invasive growth-specific transcription factor and its target genes . This function of Fus3 is essential for correct execution of the mating program and is not shared by Kss1 . We find that Kss1 controls the activity of Tec1 mainly during invasive growth by control of TEC1 gene expression . Our study suggests that signaling specificity can arise from differential regulation of a single transcription factor by two MAP kinases with shared functions in distinct developmental programs.

An Acad Bras Cienc, 2004 Dec, 76(4), 699 - 705 Epub 2004 Dec.
Inactivation of yeast inorganic pyrophosphatase by organic solvents; Grazinoli-Garrido R et al.; A number of application for enzymes in organic solvents have been developed in chemical processing, food related conversions and analyses . The only unsolved problem related to nonaqueous enzymology is the notion that enzymes in organic solvent are mostly far less active than in water . Therefore, studies concerning the mechanisms by which enzymes are inactivated by organic solvents would reveal a clear understanding of the structure-function relationship of this phenomenon . Here we analyzed the effects of a series of alcohols (methanol, ethanol, 1-propanol and 2-propanol) and acetone on the activity of yeast inorganic pyrophosphatase . We observed that solvents inactivated the enzyme in a dose-dependent manner . This inactivation is also dependent on the hydrophobicity of the solvent, where the most hydrophobic solvent is also the most effective one . The I50 for inactivation by n-alcohols are 5.9 +/- 4, 2.7 +/- 1 and 2.5 +/- 1 M for methanol, ethanol and 1-propanol, respectively . Inactivation was less effective at 37 degrees C than at 5 degrees C, when the I50 for inactivation by methanol, ethanol and 1-propanol are 4.5 +/- 2, 2.1 +/- 2 and 1.7 +/- 1 M, respectively . Our proposal is that solvent binds to the enzyme structure promoting the inactivation by stabilizing an unfolded structure, and that this binding is through the hydrophobic regions of either the protein or the solvent.

J Biol Chem . 2004 Nov 19; {Epub ahead of print}
Yeast-like mRNA capping apparatus in Giardia lamblia; Hausmann S et al.; A scheme of eukaryotic phylogeny has been suggested based on the structure and physical linkage of the RNA triphosphatase and RNA guanylyltransferase enzymes that catalyze mRNA cap formation . Here we show that the unicellular pathogen Giardia lamblia encodes an mRNA capping apparatus consisting of separate triphosphatase and guanylyltransferase components, which we characterize biochemically . We also show that native Giardia mRNAs have blocked 5' ends and that 7-methylguanosine caps promote translation of transfected mRNAs in Giardia in vivo . The Giardia triphosphatase belongs to the tunnel-family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, microsporidia, and protozoa such as Plasmodium and Trypanosoma . The tunnel enzymes adopt a unique active-site fold and are structurally and mechanistically unrelated to the cysteine-phosphatase-type RNA triphosphatases found in metazoans and plants, which comprise part of a bifunctional triphosphatase-guanylyltransferase fusion protein . All available evidence now points to the separate tunnel-type triphosphatase and guanylyltransferase as the aboriginal state of the capping apparatus . We identify a putative tunnel-type triphosphatase and a separate guanylyltransferase encoded by the red alga Cyanidioschyzon merolae . These findings place fungi, protozoa and red algae in a common lineage distinct from that of metazoa and plants.

Curr Biol, 2004 Nov 23, 14(22), 1996 - 2004
Live cell imaging of mitochondrial movement along actin cables in budding yeast; Fehrenbacher KL et al.; BACKGROUND: Mitochondrial inheritance is essential for cell division . In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria . RESULTS: We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement . In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s . Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements . In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements . CONCLUSIONS: Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast . We provide the first direct evidence that one of the proposed cargos use actin cables as tracks . In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells . Interaction of mitochondria with actin cables is required for anterograde and retrograde movement . In contrast, force generation on mitochondria is required only for anterograde movement . Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.

FEBS Lett, 2004 Nov 19, 577(3), 501 - 6
S-adenosyl-L-homocysteine hydrolase in yeast: key enzyme of methylation metabolism and coordinated regulation with phospholipid synthesis; Tehlivets O et al.; S-adenosyl-L-homocysteine hydrolase (Sah1p, EC 3.3.1.1.) is a key enzyme of methylation metabolism . It catabolizes S-adenosyl-L-homocysteine, which is formed after donation of the activated methyl group of S-adenosyl-L-methionine (AdoMet) to an acceptor, and which acts as strong competitive inhibitor of all AdoMet-dependent methyltransferases . Sah1p is an essential enzyme in yeast and one of the most highly conserved proteins with up to 80% sequence homology throughout all kingdoms of life . SAH1 expression in yeast is subject to the general transcriptional control of phospholipid synthesis . Profound changes in cellular lipid composition upon depletion of Sah1p support the notion of a tight interaction between lipid metabolism and Sah1p function.

FEBS Lett, 2004 Nov 19, 577(3), 491 - 5
Atg21p is essential for macropexophagy and microautophagy in the yeast Hansenula polymorpha; Leao-Helder AN et al.; ATG genes are required for autophagy-related processes that transport proteins/organelles destined for proteolytic degradation to the vacuole . Here, we describe the identification and characterisation of the Hansenula polymorpha ATG21 gene . Its gene product Hp-Atg21p, fused to eGFP, had a dual location in the cytosol and in peri-vacuolar dots . We demonstrate that Hp-Atg21p is essential for two separate modes of peroxisome degradation, namely glucose-induced macropexophagy and nitrogen limitation-induced microautophagy . In atg21 cells subjected to macropexophagy conditions, sequestration of peroxisomes tagged for degradation is initiated but fails to complete.

Anal Biochem, 2004 Dec 15, 335(2), 253 - 9
A yeast two-hybrid technology-based system for the discovery of PPARgamma agonist and antagonist; Chen Q et al.; Peroxisome proliferator-activated receptor gamma (PPARgamma) is an important therapeutic drug target against several diseases such as diabetes, inflammation, dyslipidemia, hypertension, and cancer . Ligand binding to PPARgamma is responsible for controlling the biological functions, and developing new technology to measure ligand-PPARgamma binding is significant for both the function study of the receptor and ligand discovery . In this study, we exploited an efficient approach for the discovery of PPARgamma agonist and antagonist via a yeast two-hybrid system based on the fact that PPARgamma interacts with the coactivator CBP (CREP-binding protein) ligand-dependently . We employed the MEL1 reporter gene instead of the traditionally used LacZ gene to evaluate the protein-protein interactions by conducting a convenient alpha-galactosidase assay in the yeast strain AH109 with genes of PPARgamma-LBD (ligand-binding domain) and CBP N terminus introduced . With this built screening platform, the EC(50) values of the PPARgamma agonists rosiglitazone, troglitazone, pioglitazone, indomethacin, 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)), and GI262570 were investigated, and the quantitatively antagonistic effect by IC(50) of the PPARgamma typical antagonist GW9662 on the rosiglitazone agonistic activity was fully examined . The reliability of this presented system evaluated by the comparable agreement of EC(50) and IC(50) values for the test compounds with the reported ones indicated that this yeast two-hybrid-based approach is powerful for PPARgamma agonist and antagonist screening . In addition, because this screening system is designed for use in a microtiter plate format where numerous chemicals could be readily screened, it is hoped that this yeast two-hybrid screening approach may be adaptable for high-throughput settings.

Biochimie, 2004 Sep-Oct, 86(9-10), 617 - 23
The Paris-Sud yeast structural genomics pilot-project: from structure to function; Quevillon-Cheruel S et al.; We present here the outlines and results from our yeast structural genomics (YSG) pilot-project . A lab-scale platform for the systematic production and structure determination is presented . In order to validate this approach, 250 non-membrane proteins of unknown structure were targeted . Strategies and final statistics are evaluated . We finally discuss the opportunity of structural genomics programs to contribute to functional biochemical annotation.

FEMS Yeast Res, 2004 Dec, 5(3), 205 - 11
Ability of human CDC25B phosphatase splice variants to replace the function of the fission yeast Cdc25 cell cycle regulator; Lemaire M et al.; CDC25 phosphatases are essential and evolutionary-conserved actors of the eukaryotic cell cycle control . To examine and compare the properties of three splicing variants of human CDC25B, recombinant fission yeast strains expressing the human proteins in place of the endogenous Cdc25 were generated and characterized . We report, that the three CDC25B variants: (i) efficiently replace the yeast counterpart in vegetative growth, (ii) partly restore the gamma and UV radiation DNA damage-activated checkpoint, (iii) fail to restore the DNA replication checkpoint activated by hydroxyurea . Although these yeast strains do not reveal the specific functions of the human CDC25B variants, they should provide useful screening tools for the identification of new cell cycle regulators and pharmacological inhibitors of CDC25 phosphatase.

Comp Biochem Physiol C Toxicol Pharmacol, 2004 Oct, 139(1-3), 147 - 52
Survey of estrogenic activity in fish feed by yeast estrogen-screen assay; Matsumoto T et al.; Fishes have been used as laboratory animal for research of estrogenic endocrine disrupters by many researchers . However, much less attention was paid to the possibility that compounds with estrogenic activity are present in fish diets . In order to examine this possibility, we measured the estrogenic activity in commercial fish feed by in vitro yeast estrogen-screen (YES) assay based on the binding ability of tested compounds to estrogen receptors . Estrogenic activity was detected in all the commercial fish feed examined (0.2-6.2 ng estradiol equivalent/g fish feed), some phytoestrogens (genistein, formononetin, equol and coumestrol; relative activity to estradiol, 8.6 x 10(-6)-1.1 x 10(-4) by giving a value of 1.0 to estradiol) and some androgens (testosterone, 11-ketotestosterone and 5 alpha-dihydrotestosterone; relative activity to estradiol, 3.0 x 10(-6)-1.2 x 10(-4)) . Therefore, it is possible that these compounds could affect the results of in vivo estrogen assay, such as vitellogenin production in male fish, especially when fish are fed commercial feed.

Curr Opin Microbiol, 2004 Dec, 7(6), 673 - 9
Aging and genetic instability in yeast; McMurray MA et al.; There is a striking link between increasing age and the incidence of cancer in humans . One of the hallmarks of cancer, genomic instability, has been observed in all types of organisms . In the yeast Saccharomyces cerevisiae, it was recently discovered that during the replicative lifespan, aging cells switch to a state of high genomic instability that persists until they die . In considering these and other recent results, we suggest that accumulation of oxidatively damaged protein in aging cells results in the loss of function of gene products critical for maintaining genome integrity . Determining the identity of these proteins and how they become damaged represents a new challenge for understanding the relationship between age and genetic instability.

Curr Opin Microbiol, 2004 Dec, 7(6), 655 - 60
Apoptosis in yeast; Madeo F et al.; Apoptosis is a highly regulated cellular suicide program crucial for metazoan development . However, dysfunction of apoptosis also leads to several diseases . Yeast undergoes apoptosis after application of acetic acid, sugar- or salt-stress, plant antifungal peptides, or hydrogen peroxide . Oxygen radicals seem to be key elements of apoptotic execution, conserved during evolution . Furthermore, several yeast orthologues of central metazoan apoptotic regulators have been identified, such as a caspase and a caspase-regulating serine protease . In addition, physiological occurrence of cell death has been detected during aging and mating in yeast . The finding of apoptosis in yeast, other fungi and parasites is not only of great medical relevance but will also help to understand some of the still unknown molecular mechanisms at the core of apoptotic execution.

Curr Opin Microbiol, 2004 Dec, 7(6), 624 - 30
Sense and sensibility: nutritional response and signal integration in yeast; Schneper L et al.; Yeast cells respond to the quantity and quality of carbon and nitrogen sources in the environment both by adjusting their transcriptional and metabolic profiles to make optimum use of the available nutrients and by selecting a developmental program--budding, pseudohyphal differentiation, quiescence or sporulation--that maximizes their potential for survival under the existing nutrient conditions . Recent studies fueled by genomic tools have refined our knowledge of the components and connections within individual pathways and the interconnections between pathways . More significantly, these studies begin to paint an as yet inchoate portrait of the yeast cells' means of processing its environmental information, in which specific transcription factors and chromatin modifying activities coordinate input from several signaling pathways to yield an appropriate and coherent response of genes involved in mass accumulation and metabolism.

Curr Opin Microbiol, 2004 Dec, 7(6), 617 - 23
Integrative studies put cell wall synthesis on the yeast functional map; Firon A et al.; The fungal cell wall field, traditionally focused on polysaccharide composition and synthesis, retains a certain static architectural imagery of structural rigidity and integrity, with the wall offering protection from a harsh environment . This picture of the wall is increasingly changing to that of a bustling construction site, as research uncovers the organizational complexity of its assembly . With recent molecular and genomic studies on Saccharomyces cerevisiae, cell wall synthesis and biology appear increasingly to be dynamic and adaptable processes that are fully integrated with the underlying cytoskeletal and polarity machinery that drive cell cycle progression.

Biochem Biophys Res Commun, 2004 Dec 24, 325(4), 1424 - 32
Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast; Bamps S et al.; The Mcs6 CDK together with its cognate cyclin Mcs2 represents the CDK-activating kinase (CAK) of fission yeast Cdc2 . We have attempted to determine complexes in which Mcs6 and Mcs2 mediate this and possible other functions . Here we characterize a novel interaction between Mcs2 and Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase . Furthermore, we identify a novel protein termed Pmh1 through its association with Skp1 . Pmh1 associates with the Mcs6-Mcs2 complex, enhancing its kinase activity, and represents the apparent homolog of metazoan Mat1 . Association of Mcs2 or Pmh1 with Skp1 does not appear to be involved in proteolytic degradation, as these complexes do not contain Pcu1, and levels of Mcs2 or Pmh1 are not sensitive to inhibition of SCF and the 26S proteasome . The identified interactions between Skp1 and two regulatory CAK subunits may reflect a novel mechanism to modulate activity and specificity of the Mcs6 kinase.

Zhonghua Liu Xing Bing Xue Za Zhi, 2004 Sep, 25(9), 805 - 7
{Application of exponent curve model to study the hepatitis B DNA recombinant yeast derived vaccine antibody levels.}; Luo FJ et al.; OBJECTIVE: To introduce exponent curve model methods in the study of the hepatitis B vaccine antibody level . METHODS: After the China made vaccine of hepatitis B DNA recombinant yeast derived vaccine (YDV) had been carried out for 5 years, data on the anti-HBsAg's titer were used to construct an exponent curve model . When the vaccination program had been carried out for 8 years, the predicating results of the model were further tested by observed number . RESULTS: The exponent curve model was Y = 165.67 exp (-0.019X) and the R(2) was 0.98 . After 8 years, the practical observed number became 35 mIU/ml, and the predicating result of the model was 27 mIU/ml, 8 mIU/ml lower than the observed number . When the vaccine had been carried out for 12 years, the predicating results of the model became 10.74 mIU/ml, still higher than 10 mIU/ml but was still in the effective range . CONCLUSION: An exponent curve model could be constructed, as long as the data of the antibody's titer was in accordance with the tendency of exponent curve . The model could be used to predict the persistence lever of vaccine antibody under certain conditions . The results showed that after 8 years, the predicting results of the model were reliably lower than the observed number.

J Chromatogr A, 2004 Oct 29, 1054(1-2), 303 - 12
Chromatographic speciation of anionic and neutral selenium compounds in Se-accumulating Brassica juncea (Indian mustard) and in selenized yeast; Kahakachchi C et al.; Selenium-accumulating plants such as Brassica juncea (Indian mustard) concentrate the element in plant shoots and roots . Such behavior may provide a cost-effective technology to clean up contaminated soils and waters that pose major environmental and human health problems (phytoremediation) . Such ability to transform selenium into bioactive compounds has important implications for human nutrition and health . Element selective characterization of B . juncea grown in the presence of inorganic selenium under hydroponic conditions provides valuable information to better understand selenium metabolism in plants . The present work determines both previously observed organoselenium species such as selenomethionine and Se-methylselenocysteine and for the first time detects the newly characterized S-(methylseleno)cysteine in plant shoots and roots when grown in the presence of selenate or selenite as the only selenium source . A key feature of this study is the complementary role of selenium and sulfur specific chromatographic detection by HPLC with interfaced inductively coupled plasma mass spectrometry (ICP-MS) detection and by derivatization GC with interfaced atomic spectral emission . HPLC-ICP-MS limits of detection for such species were in the range 5-50 ng Se mL(-1) in the injected extracts . Speciation profiles are compared with those of selenium-enriched yeast by both HPLC-ICP-MS and GC-AED.

Curr Genet, 2005 Jan, 47(1), 18 - 28 Epub 2004 Nov 18.
The yeast VPS genes affect telomere length regulation; Rog O et al.; Eukaryotic cells invest a large proportion of their genome in maintaining telomere length homeostasis . Among the 173 non-essential yeast genes found to affect telomere length, a large proportion is involved in vacuolar traffic . When mutated, these vacuolar protein-sorting (VPS) genes lead to telomeres shorter than those observed in the wild type . Using genetic analysis, we characterized the pathway by which VPS15, VPS34, VPS22, VPS23 and VPS28 affect the telomeres . Our results indicate that these VPS genes affect telomere length through a single pathway and that this effect requires the activity of telomerase and the Ku heterodimer, but not the activity of Tel1p or Rif2p . We present models to explain the link between vacuolar traffic and telomere length homeostasis.

Science, 2004 Nov 19, 306(5700), 1367 - 70
Very low gene duplication rate in the yeast genome; Gao LZ et al.; The gene duplication rate in the yeast genome is estimated without assuming the molecular clock model to be approximately 0.01 to 0.06 per gene per billion years; this rate is two orders of magnitude lower than a previous estimate based on the molecular clock model . This difference is explained by extensive concerted evolution via gene conversion between duplicated genes, which violates the assumption of the molecular clock in the analyses of duplicated genes . The average length of the period of concerted evolution and the gene conversion rate are estimated to be approximately 25 million years and approximately 28 times the mutation rate, respectively.

J Negat Results Biomed . 2004 Nov 17;3(1):5.
CP-31398, a putative p53-stabilizing molecule tested in mammalian cells and in yeast for its effects on p53 transcriptional activity; Tanner S et al.; BACKGROUND: CP-31398 is a small molecule that has been reported to stabilize the DNA-binding core domain of the human tumor suppressor protein p53 in vitro . The compound was also reported to function as a potential anti-cancer drug by rescuing the DNA-binding activity and, consequently, the transcription activation function of mutant p53 protein in mammalian tissue culture cells and in mice . RESULTS: We performed a series of gene expression experiments to test the activity of CP-31398 in yeast and in human cell cultures . With these cell-based assays, we were unable to detect any specific stimulation of mutant p53 activity by this compound . Concentrations of CP-31398 that were reported to be active in the published work were highly toxic to the human H1299 lung carcinoma and Saos-2 cell lines in our experiments . CONCLUSION: In our experiments, the small molecule CP-31398 was unable to reactivate mutant p53 protein . The results of our in vivo experiments are in agreement with the recently published biochemical analysis of CP-31398 showing that this molecule does not bind p53 as previously claimed, but intercalates into DNA.

Biogerontology, 2004, 5(5), 305 - 16
The histone acetyltransferase GCN5 modulates the retrograde response and genome stability determining yeast longevity; Kim S et al.; Transcriptional silencing decreases at both subtelomeric and silent mating-type loci and increases at the ribosomal DNA locus during the replicative life span of the yeast Saccharomyces cerevisiae . Evidence exists that epigenetic changes in the regulatory state of chromatin may be a causal factor in determining yeast longevity and that histone deacetylases play a role . The significance of histone acetylation has been examined here in more detail . Deletion of the histone acetyltransferase gene GCN5 suppressed the extension of replicative life span afforded by the induction of the retrograde response, which signals mitochondrial dysfunction and leads to changes in nuclear gene expression . It was difficult to ascribe this effect to changes in transcriptional silencing in any of the three known types of heterochromatin . However, a promoter related effect was uncovered by the participation of GCN5 in the induction of the retrograde response . Gcn5p and the retrograde signal transducer Rtg2p are components of the histone acetyltransferase coactivator complex SLIK . Rtg2p blocks the production of extrachromosomal ribosomal DNA circles when it is not engaged in transmission of the retrograde signal . Deletion of GCN5 , which disrupts the integrity of SLIK, suppressed circle accumulation . The results indicate that Gcn5p and SLIK impact the interplay between the retrograde response signal and Rtg2p with consequences for the induction of the response and circle production . Rtg2p and Gcn5p in the SLIK complex link metabolism to stress responses, chromatin-dependent gene regulation, and genome stability in yeast aging.

RNA, 2004 Dec, 10(12), 1907 - 15
Nonsense-mediated decay does not occur within the yeast nucleus; Kuperwasser N et al.; Nonsense-mediated decay (NMD) is a eukaryotic regulatory process that degrades mRNAs with premature termination codons (PTCs) . Although NMD is a translation-dependent process, there is evidence from mammalian systems that PTC recognition and mRNA degradation takes place in association with nuclei . Consistent with this notion, degradation of mammalian PTC-containing mRNAs occurs when they are bound by the cap binding complex (CBC) during a "pioneer" round of translation . Moreover, there are reports indicating that a PTC can trigger other nuclear events such as alternative splicing, abnormal 3' end processing, and accumulation of pre-mRNA at transcription sites . To examine whether a PTC can elicit similar nuclear events in yeast, we used RNA export-defective mutants to sequester mRNAs within nuclei . The results indicate that nuclear PTC-containing yeast RNAs are NMD insensitive . We also observed by fluorescent in situ hybridization that there was no PTC effect on mRNA accumulated at the site of transcription . Finally, we show that yeast NMD occurs minimally if at all on CBC-bound transcripts, arguing against a CBC-mediated pioneer round of translation in yeast . The data taken together indicate that there are no direct consequences of a PTC within the yeast nucleus.

RNA, 2004 Dec, 10(12), 1888 - 99
A synthetic A tail rescues yeast nuclear accumulation of a ribozyme-terminated transcript; Dower K et al.; To investigate the role of 3' end formation in yeast mRNA export, we replaced the mRNA cleavage and polyadenylation signal with a self-cleaving hammerhead ribozyme element . The resulting RNA is unadenylated and accumulates near its site of synthesis . Nonetheless, a significant fraction of this RNA reaches the cytoplasm . Nuclear accumulation was relieved by insertion of a stretch of DNA-encoded adenosine residues immediately upstream of the ribozyme element (a synthetic A tail) . This indicates that a 3' stretch of adenosines can promote export, independently of cleavage and polyadenylation . We further show that a synthetic A tail-containing RNA is unaffected in 3' end formation mutant strains, in which a normally cleaved and polyadenylated RNA accumulates within nuclei . Our results support a model in which a polyA tail contributes to efficient mRNA progression away from the gene, most likely through the action of the yeast polyA-tail binding protein Pab1p.

Mol Cell, 2004 Nov 19, 16(4), 619 - 30
An SMC-domain protein in fission yeast links telomeres to the meiotic centrosome; Flory MR et al.; Abnormal centrosomal structures similar to those occurring in human cancers are induced in fission yeast by overexpression of the pericentrin homolog Pcp1p . Analysis of abnormal Pcp1p-containing structures with quantitative mass spectrometry and isotope-coded affinity tags identified a coiled-coil, structural maintenance of chromosomes (SMC) domain protein . This protein, termed Ccq1p (coiled-coil protein quantitatively enriched), localizes with Taz1p to telomeres in normal vegetative cells . Fluorescence resonance energy transfer (FRET) measurements indicate that Ccq1p also interacts with centrosomal Pcp1p in mating pheromone-stimulated cells containing centrosomally clustered telomeres . We provide evidence that the Ccq1p-Pcp1p interaction, while essential for meiosis, is deleterious when forced to occur during vegetative growth . Cells lacking one ccq1 allele exhibit a loss-of-function phenotype including abnormally long cell length, chromosome segregation failure, telomeric shortening, and defective telomeric clustering during meiotic prophase . Our data indicate a mechanism underlying meiotic chromosomal bouquet formation and suggest a recruitment model for supernumerary centrosome toxicity.

Theor Biol Med Model . 2004 Nov 16;1(1):12.
Size control in growing yeast and mammalian cells; Sveiczer A et al.; BACKGROUND: In a recent publication it was claimed that cultured mammalian cells, in contrast to yeasts, maintain a constant size distribution in the population without a size checkpoint . This inference may be challengeable . RESULTS: (1) It is argued that "weak" size control implies the existence of a checkpoint, and unfortunately the technique used by Conlon and Raff might obscure such a weak mechanism . (2) Previous investigations of size control in yeasts have shown that individual cell data, rather than means and variances of cell populations, are prerequisites for reliable interpretation . (3) No experimental data so far obtained suggest that in any cell culture a linear growth pattern in cell mass can maintain size homeostasis on its own without size control . (4) Studies on fission yeast mutants indicate that the molecular mechanisms of size control vary with genetic background, implying that no single mechanism is likely to apply to any cell type, including cultured mammalian cells, under all conditions . CONCLUSION: The claim that cultured mammalian cells maintain size homeostasis without a checkpoint needs to be re-evaluated by measurements on individual cells.

Genetics . 2004 Nov 15; {Epub ahead of print}
Genetic Factors That Regulate the Attenuation of the General Stress Response of Yeast; Bose S et al.; The general stress response of the yeast involves the induction of approximately two hundred genes in response to any one of several stresses . These genes are activated by Msn2 and repressed by the Srb10 kinase, a member of the mediator complex . Normally, Msn2 is exported from the nucleus, and Srb10 represses STRE gene expression . Under stress, Msn2 re-localizes to the nucleus, and, with the relief of Srb10 repression, activates transcription . The stress response is rapid, but quickly attenuated . We show here this attenuation is due to a nuclear-dependent degradation of Msn2 . Msn2 rapidly disappeared from cells after heat or osmotic shock . This disappearance was not due to a change in MSN2 RNA levels which remain constant during stress . Pulse-chase experiments confirmed the stress-dependent Msn2 degradation . The levels of Msn2 were significantly reduced in msn5 deletion cells which have been shown to constitutively retain Msn2 in the nucleus . The degradation was Srb10-dependent; Msn2 was not degraded in an srb10 deletion mutant . An Msn2 internal deletion mutant was insensitive to Srb10 repression, but was degraded by the Srb10-dependent mechanism . Thus, this mutant uncoupled Srb10 repression from degradation.

Nucleic Acids Res, 2004 Nov 15, 32(20), 5981 - 90 Print 2004.
Roles of Rad23 protein in yeast nucleotide excision repair; Xie Z et al.; Nucleotide excision repair (NER) removes many different types of DNA lesions . Most NER proteins are indispensable for repair . In contrast, the yeast Rad23 represents a class of accessory NER proteins, without which NER activity is reduced but not eliminated . In mammals, the complex of HR23B (Rad23 homolog) and XPC (yeast Rad4 homolog) has been suggested to function in the damage recognition step of NER . However, the precise function of Rad23 or HR23B in NER remains unknown . Recently, it was suggested that the primary function of RAD23 protein in NER is its stabilization of XPC protein . Here, we tested the significance of Rad23-mediated Rad4 stabilization in NER, and analyzed the repair and biochemical activities of purified yeast Rad23 protein . Cellular Rad4 was indeed stabilized by Rad23 in the absence of DNA damage . Persistent overexpression of Rad4 in rad23 mutant cells, however, largely failed to complement the ultraviolet sensitivity of the mutant . Consistently, deficient NER in rad23 mutant cell extracts could not be complemented by purified Rad4 protein in vitro . In contrast, partial complementation was observed with purified Rad23 protein . Specific complementation to the level of wild-type repair was achieved by adding purified Rad23 together with small amounts of Rad4 protein to rad23 mutant cell extracts . Purified Rad23 protein was unable to bind to DNA, but stimulated the binding activity of purified Rad4 protein to N-acetyl-2-aminofluorene-damaged DNA . These results support two roles of Rad23 protein in NER: (i) its direct participation in the repair biochemistry, possibly due to its stimulatory activity on Rad4-mediated damage binding/recognition; and (ii) its stabilization of cellular Rad4 protein.

Proc Natl Acad Sci U S A, 2004 Nov 23, 101(47), 16495 - 500 Epub 2004 Nov 23.
Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques; Bystricky K et al.; Little is known about how chromatin folds in its native state . Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast . Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes . We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14-100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives . The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170-220 nm and a mass density of approximately 110-150 bp/nm . These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure . Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry . The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2431 - 5
Kazachstania aerobia sp . nov., an ascomycetous yeast species from aerobically deteriorating corn silage; Lu HZ et al.; In an investigation of the yeast biota involved in silage deterioration, a considerable number of strains belonging to Saccharomyces and related genera were isolated from aerobically deteriorating corn silage in Tochigi, Japan . Analysis of sequences of the internal transcribed spacer and the large-subunit rRNA gene D1/D2 domain and electrophoretic karyotyping indicated that two of the strains, NS 14(T) and NS 26, represent a novel species with close phylogenetic relationships to Kazachstania servazzii and Kazachstania unispora . It is proposed that the novel species be named Kazachstania aerobia sp . nov., with NS 14(T) (=AS 2.2384(T)=CBS 9918(T)) as the type strain.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2409 - 29
Expansion of the Candida tanzawaensis yeast clade: 16 novel Candida species from basidiocarp-feeding beetles; Suh SO et al.; A major clade of new yeast taxa from the digestive tract of basidiocarp-feeding beetles is recognized based on rRNA gene sequence analyses . Almost 30 % of 650 gut isolates formed a statistically well-supported clade that included Candida tanzawaensis . The yeasts in the clade were isolated from 11 families of beetles, of which Tenebrionidae and Erotylidae were most commonly sampled . Repeated isolation of certain yeasts from the same beetle species at different times and places indicated strong host associations . Sexual reproduction was never observed in the yeasts . Based on comparisons of small- and large-subunit rRNA gene sequences and morphological and physiological traits, the yeasts were placed in Candida ambrosiae and in 16 other undescribed taxa . In this report, the novel species in the genus Candida are described and their relationships with other taxa in the Saccharomycetes are discussed . The novel species and their type strains are as follows: Candida guaymorum (NRRL Y-27568(T)=CBS 9823(T)), Candida bokatorum (NRRL Y-27571(T)=CBS 9824(T)), Candida kunorum (NRRL Y-27580(T)=CBS 9825(T)), Candida terraborum (NRRL Y-27573(T)=CBS 9826(T)), Candida emberorum (NRRL Y-27606(T)=CBS 9827(T)), Candida wounanorum (NRRL Y-27574(T)=CBS 9828(T)), Candida yuchorum (NRRL Y-27569(T)=CBS 9829(T)), Candida chickasaworum (NRRL Y-27566(T)=CBS 9830(T)), Candida choctaworum (NRRL Y-27584(T)=CBS 9831(T)), Candida bolitotheri (NRRL Y-27587(T)=CBS 9832(T)), Candida atakaporum (NRRL Y-27570(T)=CBS 9833(T)), Candida panamericana (NRRL Y-27567(T)=CBS 9834(T)), Candida bribrorum (NRRL Y-27572(T)=CBS 9835(T)), Candida maxii (NRRL Y-27588(T)=CBS 9836(T)), Candida anneliseae (NRRL Y-27563(T)=CBS 9837(T)) and Candida taliae (NRRL Y-27589(T)=CBS 9838(T)).

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2405 - 8
Candida leandrae sp . nov., an asexual ascomycetous yeast species isolated from tropical plants; Ruivo CC et al.; The novel yeast species Candida leandrae is described based on eight isolates from decaying fruits of Leandra reversa Cogn . (Melastomataceae) in an Atlantic rainforest site in Brazil, one from a Convolvulaceae flower in Costa Rica and one from a drosophilid in Hawai'i . The strains differed in their colony morphology, one being butyrous and smooth and the other being filamentous and rugose . Sequences of the D1/D2 domains of the large-subunit rRNA gene from both morphotypes were identical . C . leandrae belongs to the Kodamaea clade and is closely related to Candida restingae . The two species can be separated on the basis of growth at 37 degrees C and the assimilation of melezitose, negative in the novel species . The type culture of C . leandrae is strain UNESP 00-64R(T) (=CBS 9735(T)=NRRL Y-27757(T)).

J Dairy Sci, 2004 Dec, 87(12), 4178 - 81
Feed efficiency of mid-lactation dairy cows fed yeast culture during summer; Schingoethe DJ et al.; Thirty-eight Holstein cows (26 multiparous and 12 primiparous), that averaged 105 d postpartum at the start of the experiment, were used to evaluate the feeding of yeast culture (60 g/cow daily of Diamond V XP) on production efficiency during hot summer weather . From early June until early September and after a 2-wk covariate period, cows were fed a control diet without or with 60 g of yeast culture/cow daily for 12 wk . Weekly daytime high temperatures in the free-stall barn during the 12-wk period averaged 33 degrees C (28 to 39 degrees C) . Total mixed diets on a dry matter (DM) basis consisted of corn silage (28%), alfalfa hay (21%), and a concentrate mix (51%) without or with the yeast culture added to the total mixed ration at the time of feeding . Milk production (34.9 and 35.4 kg/d, for control and yeast culture treatment, respectively), 4% fat-corrected milk (31.2 and 32.0 kg/d), energy-corrected milk (ECM; 33.4 and 34.2 kg/d), and DM intake (23.1 and 22.1 kg/d) were similar for cows fed control and yeast culture diets . Percentages of milk fat (3.34 and 3.41) and true protein (2.85 and 2.87) were similar for both diets . Feed efficiency defined as kilogram of ECM/kilogram of DM intake was improved by 7% for cows fed the yeast culture . Body weights and body condition scores were similar for both groups . The results suggest that the yeast culture can improve feed efficiency of heat stressed dairy cows in midlactation.

J Mol Biol, 2004 Dec 3, 344(4), 1071 - 87
Solution structure of the dimeric SAM domain of MAPKKK Ste11 and its interactions with the adaptor protein Ste50 from the budding yeast: implications for Ste11 activation and signal transmission through the Ste50-Ste11 complex; Bhattacharjya S et al.; Ste11, a homologue of mammalian MAPKKKs, together with its binding partner Ste50 works in a number of MAPK signaling pathways of Saccharomyces cerevisiae . Ste11/Ste50 binding is mediated by their sterile alpha motifs or SAM domains, of which homologues are also found in many other intracellular signaling and regulatory proteins . Here, we present the solution structure of the SAM domain or residues D37-R104 of Ste11 and its interactions with the cognate SAM domain-containing region of Ste50, residues M27-Q131 . NMR pulse-field-gradient (PFG) and rotational correlation time measurements (tauc) establish that the Ste11 SAM domain exists predominantly as a symmetric dimer in solution . The solution structure of the dimeric Ste11 SAM domain consists of five well-defined helices per monomer packed into a compact globular structure . The dimeric structure of the SAM domain is maintained by a novel dimer interface involving interactions between a number of hydrophobic residues situated on helix 4 and at the beginning of the C-terminal long helix (helix 5) . The dimer structure may also be stabilized by potential salt bridge interactions across the interface . NMR H/2H exchange experiments showed that binding of the Ste50 SAM to the Ste11 SAM very likely involves the positively charged extreme C-terminal region as well as exposed hydrophobic patches of the dimeric Ste11 SAM domain . The dimeric structure of the Ste11 SAM and its interactions with the Ste50 SAM may have important roles in the regulation and activation of the Ste11 kinase and signal transmission and amplifications through the Ste50-Ste11 complex.

J Mol Biol, 2004 Dec 3, 344(4), 893 - 905
Regulation of transcriptional silencing in yeast by growth temperature; Bi X et al.; Increasing evidence indicates that transcriptionally silent chromatin structure is dynamic and may change its conformation in response to external or internal stimuli . We show that growth temperature affects all three forms of transcriptional silencing in Saccharomyces cerevisiae . In general, increasing the temperature within the range of 23-37 degrees C strengthens HM and telomeric silencing but reduces rDNA silencing . High temperature (37 degrees C) can suppress the silencing defects of histone H4 mutants . We demonstrate that DNA at the silent HML locus becomes more and more negatively supercoiled as temperature increases in a Sir-dependent manner, which is indicative of enhanced silent chromatin . This enhancement of silent chromatin is not dependent on silencers and therefore does not require de novo assembly of silent chromatin . We also present evidence suggesting that MAP kinase-mediated Sir3p hyperphosphorylation, which plays a role in regulating silencing in response to certain stress conditions, is not involved in high temperature-induced strengthening of silencing . In addition, Pnc1p, a positive regulator of Sir2p activity, plays no role in thermal regulation of silencing . Therefore, growth temperature regulates transcriptional silencing by a novel mechanism.

Biochemistry, 2004 Nov 23, 43(46), 14612 - 23
Role of base stacking and sequence context in the inhibition of yeast DNA polymerase eta by pyrene nucleotide; Hwang H et al.; The Y family DNA polymerase yeast pol eta inserts pyrene deoxyribose monophosphate (dPMP) in preference to A opposite an abasic site, the 3'-T of a thymine dimer, and a normal T with almost equal efficiency . In contrast, pol A family polymerases such as Klenow fragment and T7 DNA polymerase only insert dPMP efficiently opposite an abasic site and the 3'-T of a thymine dimer but not opposite undamaged DNA . Pyrene nucleotide is also an efficient chain-terminating inhibitor of DNA synthesis by pol eta but not by Klenow fragment or T7 DNA polymerase . To better understand the origin of the efficiency and sequence specificity of dPMP insertion by pol eta, the kinetics of dPMP insertion opposite various templates have been determined . In one sequence context, the efficiency of dPMP insertion increases 4.6-fold opposite G < A << T < C, suggesting that the templating nucleotide modulates dPMP insertion efficiency by having to destack prior to dPTP binding . The efficiency of insertion of dPMP opposite T in the same sequence context increases 7-fold for primers terminating in G < A < C < T and is similar to that observed for nontemplated blunt-end extension, suggesting that stacking interactions between the pyrene and the primer terminus are also important . On heterogeneous templates, the average selectivity for dPMP insertion relative to the complementary dNMP decreases in the order of dAMP > dGMP > dTMP > dCMP, from a high of 5.8 when dAMP is to be inserted following a T to a low of 0.5 when dCMP is to be inserted following a C . The relative preference for dPMP insertion at a given site can be largely explained by the energetic cost of destacking the templating base and stacking of pyrene nucleotide relative to that of stacking and base pairing the complementary nucleotide . Thus, pyrene nucleotide represents a novel class of nucleotide-based chain-terminating DNA synthesis inhibitors whose base portion consists of a hydrophobic, non-hydrogen bonding, base-pair mimic.

Cell Death Differ, 2004 Dec, 11(12), 1309 - 16
The Caenorhabditis elegans CED-9 protein does not directly inhibit the caspase CED-3, in vitro nor in yeast; Jabbour AM et al.; A genetically defined pathway orchestrates the removal of 131 of the 1090 somatic cells generated during the development of the hermaphrodite nematode Caenorhabditis elegans . Regulation of apoptosis is highly evolutionarily conserved and the nematode cell death pathway is a valuable model for studying mammalian apoptotic pathways, the dysregulation of which can contribute to numerous diseases . The nematode caspase CED-3 is ultimately responsible for the destruction of worm cells in response to apoptotic signals, but it must first be activated by CED-4 . CED-9 inhibits programmed cell death and considerable data have demonstrated that CED-9 can directly bind and inhibit CED-4 . However, it has been suggested that CED-9 may also directly inhibit CED-3 . In this study, we used a yeast-based system and biochemical approaches to explore this second potential mechanism of action . While we confirmed the ability of CED-9 to inhibit CED-4, our data argue that CED-9 can not directly inhibit CED-3.

Mol Cell Proteomics . 2004 Nov 12; {Epub ahead of print}
A Proteomic Strategy for Gaining Insights into Protein Sumoylation in Yeast; Denison C et al.; Sumoylation represents a vital post-translational modification that pervades numerous aspects of cell biology, including protein targeting, transcriptional regulation, signal transduction and cell division . However, despite its broad reaching effects, most biological outcomes of protein sumoylation remain poorly understood . In an effort to provide further insight into this complex process, a proteomics approach was undertaken to identify the targets of sumoylation en mass . Specifically, SUMO-conjugated proteins were isolated by a double-affinity purification procedure from a Saccharomyces cerevisiae strain engineered to express tagged SUMO . The components of the isolated protein mixture were then identified by subsequent (LC-MS/MS) analysis using an LTQ FT mass spectrometer . In this manner, 159 candidate sumoylated proteins were identified by two or more peptides . Furthermore, the high accuracy of the instrument, combined with stringent search criteria, enabled the identification of an additional 92 putative candidates by only one peptide . The validity of this proteomics approach was confirmed by performing subsequent western blot experiments for numerous proteins and determining the actual sumoylation sites for several other substrates . These data combine with recent works to further our understanding of the breadth and impact of protein sumoylation in a diverse array of biological processes.

Mol Cell Biol, 2004 Dec, 24(23), 10479 - 91
Yeast shuttling SR proteins Npl3p, Gbp2p, and Hrb1p are part of the translating mRNPs, and Npl3p can function as a translational repressor; Windgassen M et al.; A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled . Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export . Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation . While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation . Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator . Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A . Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs . Together, these data shed light onto the transformation of an exporting to a translating mRNP.

Mol Cell Biol, 2004 Dec, 24(23), 10313 - 27
Activation of the DNA damage checkpoint in yeast lacking the histone chaperone anti-silencing function 1; Ramey CJ et al.; The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of genomic integrity . Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin assembly factors, including anti-silencing function 1 (ASF1) . To investigate the role of chromatin structure in the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p . We found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage checkpoint . Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and to DNA replicational stresses . Although yeast lacking Asf1p do complete DNA replication, they have greatly elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green fluorescent protein foci . The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are fully functional for double-strand DNA repair . Our data indicate that the altered chromatin structure in asf1 mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.

Mol Cell Biol, 2004 Dec, 24(23), 10180 - 92
Yeast chromatin assembly complex 1 protein excludes nonacetylatable forms of histone H4 from chromatin and the nucleus; Glowczewski L et al.; In yeast, the establishment and maintenance of a transcriptionally silent chromatin state are dependent upon the acetylation state of the N terminus of histone proteins . Histone H4 proteins that contain mutations in N-terminal lysines disrupt heterochromatin and result in yeast that cannot mate . Introduction of a wild-type copy of histone H4 restores mating, despite the presence of the mutant protein, suggesting that mutant H4 protein is either excluded from, or tolerated in, chromatin . To understand how the cell differentiates wild-type histone and mutant histone in which the four N-terminal lysines were replaced with alanine (H4-4A), we analyzed silencing, growth phenotypes, and the histone composition of chromatin in yeast strains coexpressing equal amounts of wild-type and mutant H4 proteins (histone H4 heterozygote) . We found that histone H4 heterozygotes have defects in heterochromatin silencing and growth, implying that mutations in H4 are not completely recessive . Nuclear preparations from histone H4 heterozygotes contained less mutant H4 than wild-type H4, consistent with the idea that cells exclude some of the mutant histone . Surprisingly, the N-terminal nuclear localization signal of H4-4A fused to green fluorescent protein was defective in nuclear localization, while a mutant in which the four lysines were replaced with arginine (H4-4R) appeared to have normal nuclear import, implying a role for the charged state of the acetylatable lysines in the nuclear import of histones . The biased partial exclusion of H4-4A was dependent upon Cac1p, the largest subunit of yeast chromatin assembly factor 1 (CAF-1), as well as upon the karyopherin Kap123p, but was independent of Cac2p, another CAF-1 component, and other chromatin assembly proteins (Hir3p, Nap1p, and Asf1p) . We conclude that N-terminal lysines of histone H4 are important for efficient histone nuclear import . In addition, our data support a model whereby Cac1p and Kap123 cooperate to ensure that only appropriately acetylated histone H4 proteins are imported into the nucleus.

BJU Int, 2004 Nov, 94(7), 996 - 1002
A modified yeast assay used on archival samples of localized prostate cancer tissue improves the detection of p53 abnormalities and increases their predictive value; Shi XB et al.; OBJECTIVE: To determine the frequency and predictive value of p53 mutations in localized prostate cancer, comparing the accuracy of detection using immunohistochemistry (IHC) with a modified yeast assay, on archival tissue samples . MATERIALS AND METHODS: Prostate cancer tissue was obtained from 98 patients who had >/= 2 years of clinical follow-up after radical prostatectomy . DNA sequencing was used to verify the presence of p53 mutations in samples that were immunopositive or that gave evidence for p53 alterations using the yeast assay . The IHC and yeast findings were compared with patient outcome to determine the predictive value of these two test types . RESULTS: Fifty-five tumours (57%) were immunopositive, and 58 (59%) were positive using the yeast assay . Sequence-confirmed p53 mutations occurred in 44 (45%) cases . The IHC protocol generated 49% (27/55) false-positive and 36% (15/42) false-negative results, and was 65% sensitive and 50% specific, with an overall accuracy of 57% . The yeast assay resulted in 24% (14/58) false-positive results with a specificity of 74% and an accuracy of 86% . When the p53 status of these patients was correlated with their clinical outcome, patients who had sequence-confirmed p53 mutations had a 2.6-fold greater failure rate (P = 0.026) and a 2.5-fold greater risk of dying from prostate cancer (P = 0.05) . Notably, mutations in exon 6 predicted a six-fold increase in treatment failure (P = 0.043) and a 5.3-fold increase in the chance of dying from prostate cancer (P = 0.009) . Abnormal yeast-assay findings gave similar predictive results to those obtained for DNA sequencing, while immunopositivity did not correspond to patient outcome . CONCLUSIONS: Mutations of p53 occurred in 45% of localized prostate cancers . These alterations have important prognostic implications . The yeast assay was more accurate for detecting p53 mutations than the IHC protocol used and, unlike IHC, the results of the yeast assay were predictive of patient outcome.

EMBO Rep, 2004 Dec, 5(12), 1148 - 52
Crystal structure of yeast V-ATPase subunit C reveals its stator function; Drory O et al.; Vacuolar H(+)-ATPase (V-ATPase) has a crucial role in the vacuolar system of eukaryotic cells . It provides most of the energy required for transport systems that utilize the proton-motive force that is generated by ATP hydrolysis . Some, but not all, of the V-ATPase subunits are homologous to those of F-ATPase and the nonhomologous subunits determine the unique features of V-ATPase . We determined the crystal structure of V-ATPase subunit C (Vma5p), which does not show any homology with F-ATPase subunits, at 1.75 A resolution . The structural features suggest that subunit C functions as a flexible stator that holds together the catalytic and membrane sectors of the enzyme . A second crystal form that was solved at 2.9 A resolution supports the flexible nature of subunit C . These structures provide a framework for exploring the unique mechanistic features of V-ATPases.

J Bioenerg Biomembr, 2004 Oct, 36(5), 429 - 38
Analysis of the secondary structure of the cys-less yeast mitochondrial citrate transport protein and four single-cys variants by circular dichroism; Cascio M et al.; Utilizing cysteine scanning mutagenesis, with functional Cys-less citrate transport protein (CTP) serving as the starting template, we previously demonstrated that four single-Cys mutants located in transmembrane domains III and IV, rendered the CTP nonfunctional . The present investigations assess and quantify the secondary structure of the Cys-less CTP and the four single-Cys mutants, both in the absence and presence of citrate, via circular dichroism (CD) spectroscopy . In detergent micelles, highly purified Cys-less CTP contained approximately 50% alpha-helix and approximately 20% beta-sheet . The CD spectra of the G119C, E122C, R181C, and R189C mutants in detergent micelles were virtually superimposable with that of the functional Cys-less CTP, thereby suggesting that the wild-type residues, rather than affecting structure, may assume important mechanistic roles . Exogenously added citrate caused a significant change in the CD spectra of all solubilized CTP samples . Analyses of the spectra of the Cys-less CTP indicated an approximately 10% increase in its alpha-helical content in the presence of citrate . The conformational changes effected by the addition of substrate were less pronounced with the single-Cys mutants . Studies of the Cys-less CTP reconstituted in liposomes indicated that while the CD spectra was red-shifted, the net secondary structure of the reconstituted carrier is approximately equivalent to that of the transporter in detergent micelles, and displayed a response to added citrate . In combination, the above studies indicate that purified Cys-less CTP in either sarkosyl micelles or in liposomes, and the four inactive single-Cys mutants in sarkosyl micelles, retain native-like structure, and thus represent ideal material for detailed structural characterization.

J Mol Biol, 2004 Nov 26, 344(3), 697 - 706
Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast; Stone M et al.; Conjugation of proteins to ubiquitin plays a central role for a number of cellular processes including endocytosis, DNA repair and degradation by the 26S proteasome . However, ubiquitination is reversible as a number of deubiquitinating enzymes mediate the disassembly of ubiquitin-protein conjugates . Some deubiquitinating enzymes are associated with the 26S proteasome contributing to and regulating the particle's activity . Here, we characterise fission yeast Uch2 and Ubp6, two proteasome associated deubiquitinating enzymes . The human orthologues of these enzymes are known as Uch37 and Usp14, respectively . We report that the subunit Uch2/Uch37 is the major deubiquitinating enzyme associated with the fission yeast 26S proteasome . In contrast, the activity of Ubp6 appears to play a more regulatory and/or structural role involving the proteasome subunits Mts1/Rpn9, Mts2/Rpt2 and Mts3/Rpn12, as Ubp6 becomes essential when activity of these subunits is compromised by conditional mutations . Finally, when the genes encoding Uch2/Uch37 and Ubp6 are disrupted, the cells are viable without showing obvious signs of impaired ubiquitin-dependent proteolysis, indicating that other deubiquitinating enzymes may remedy for the redundancy of these enzymes.

J Am Mosq Control Assoc, 2004 Sep, 20(3), 261 - 4
Yeast-generated CO2 as a convenient source of carbon dioxide for adult mosquito sampling; Saitoh Y et al.; A new, convenient method was developed to supply CO2 for mosquito sampling by using yeast, which converts sugar into CO2 and ethyl alcohol . The system could, at average, generate 32.4 ml/min of CO2 for at least 27 h . The total weight of the CO2 generated was estimated to be 94 g . The efficacy of yeast-generated CO2 as attractant for mosquitoes was significant, and the following 6 mosquito species were collected using yeast-generated CO2 traps from July to September 2003 in a residential area of southern and northern Yokohama City, Japan: Aedes albopictus (Skuse), Armigeres subalbatus (Coquillett), Culex halifaxii Theobald, Cx . pipiens pallens Coquillett, Ochlerotatus japonicus (Theobald), and Tripteroides bambusa (Yamada) . Besides mosquitoes, various other insects were collected in the trap . Species compositions of insects collected in yeast-generated CO2 traps and dry-ice-baited traps were compared.

Nat Struct Mol Biol, 2004 Dec, 11(12), 1198 - 205 Epub 2004 Dec.
Cell cycle-dependent regulation of yeast telomerase by Ku; Fisher TS et al.; The heterodimeric Ku complex affects telomere structure in diverse organisms . We report here that in the absence of Ku, the catalytic subunit of telomerase, Est2p, was not telomere-associated in G1 phase, and its association in late S phase was decreased . The telomere association of Est1p, a telomerase component that binds telomeres only in late S phase, was also reduced in the absence of Ku . The effects of Ku on telomerase binding require a 48-nucleotide (nt) stem-loop region of TLC1 telomerase RNA . Ku interacts with TLC1 RNA via this 48-nt region throughout the cell cycle, but this interaction was reduced after telomere replication . These data support a model in which Ku recruits telomerase to telomeres in G1 phase when telomerase is inactive and promotes telomerase-mediated telomere lengthening in late S phase.

Curr Biol, 2004 Nov 9, 14(21), 1968 - 72
Proteolysis contributes to the exclusive centromere localization of the yeast Cse4/CENP-A histone H3 variant; Collins KA et al.; Kinetochores are the specialized protein structures that form on centromeric DNA and direct chromosome segregation . It is critical that all chromosomes assemble a single kinetochore every cell cycle . One hallmark of all eukaryotic kinetochores is CENP-A, an essential centromeric histone H3 (CenH3) variant . Overexpression of CENP-A causes mislocalization to euchromatin, which could lead to deleterious consequences because CENP-A overexpression is associated with colorectal cancer . Although CENP-A protein levels are important for genomic stability, little is known about the mechanisms of CenH3 regulation . Here, we show that the levels of the budding yeast CenH3, Cse4, are regulated by ubiquitin-proteasome-mediated proteolysis . Because mutation of all Cse4 lysine residues did not completely stabilize the protein, we isolated a dominant lethal mutant, CSE4-351, that was stable . The Cse4-351 protein localized to euchromatin, suggesting that proteolysis prevents CenH3 euchromatic localization . When wild-type Cse4 was fused to a degron signal, the soluble Cse4 protein was rapidly degraded, but the centromere bound Cse4 was stable, indicating that centromere localization protects Cse4 from degradation . Taken together, these data identify proteolysis as one mechanism that contributes to the restricted centromere localization of the yeast CenH3.

Cell Mol Biol (Noisy-le-grand), 2004 Jun, 50(4), 437 - 45
Piezophysiology of yeast: occurrence and significance; Abe F; Hydrostatic pressure is a thermodynamic parameter that has recently received further consideration in various experimental fields . Although the physicochemical basis of pressure effects is well established, the effects of high pressure on in vivo biological processes have not been systematically investigated due to its presumed complexity . The word "piezophysiology" was proposed to describe the unique cellular responses to high pressure in living cells . The yeast Saccharomyces cerevisiae is one of the best-characterized organisms in many fields in bioscience . Here I review the accumulated literature on cellular responses to increasing hydrostatic pressure in yeast, focusing on survival, growth, gene expression and metabolism.

Cell Mol Biol (Noisy-le-grand), 2004 Jun, 50(4), 317 - 22
Pressure-temperature-induced denaturation of yeast phosphoglycerate kinase, a double-domain protein; Tanaka T et al.; Pressure-induced denaturation of yeast phosphoglycerate kinase was studied at various temperatures, as a model double-domain protein, using intrinsic fluorescence, 4th derivative absorbance, CD, and DSC . A thermodynamic transition intermediate was observed in the pressure-denaturation, as was reported for the cold denaturation . From the different response of Trp and Tyr residues, as monitored by fluorescence and 4th derivative absorbance changes, the C-terminal domain carrying all the Trp residues seemed to exert structural changes at relatively lower pressure . A further structural change involving both domains was observed at higher pressures . The two-step changes occurred almost simultaneously during heat denaturation.

Gene, 2004 Nov 24, 342(2), 211 - 8
Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments; Blaise L et al.; Yeast display is a powerful technology for the affinity maturation of human antibody fragments . However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10(6) and 10(7) are typically possible . We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae {van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R.C., Hoogenboom, H.R., Hufton, S.E., 2003 . Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries . FEBS Lett . 546, 288-294} . This discovery and the knowledge that Fab antibodies are heterodimeric suggest that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type . These separate repertoires can then be combined by highly efficient yeast mating . Using this approach, we have rapidly generated a naive human Fab yeast display library of over 10(9) clones . In addition, utilizing error-prone polymerase chain reaction, we have diversified Fab sequences and generated combinatorial and hierarchical chain shuffled libraries with complexities of up to 5 x 10(9) clones . These libraries have been selected for higher affinity using a repeating process of mating-driven chain shuffling and flow cytometric sorting.

FEBS Lett, 2004 Nov 5, 577(1-2), 215 - 9
The HECT E3 ubiquitin ligase Rsp5 is important for ubiquitin homeostasis in yeast; Krsmanovic T et al.; The HECT E3 ubiquitin ligase Rsp5, a yeast member of the Nedd4 family, has been implicated in many different aspects of cell physiology . Here, we present evidence that Rsp5 function is important for ubiquitin homeostasis . Several observations suggest that ubiquitin is limiting in the rsp5-1 mutant . Reduced synthesis of ubiquitin appears to contribute to ubiquitin depletion . A transient inhibition of general protein synthesis is observed in a wildtype strain upon heat-shock . While the wildtype cells quickly recover from this transient arrest, the rsp5-1 cells remain arrested . This suggests that Rsp5 is important for recovery from heat-induced protein synthesis arrest . Our results suggest that rsp5 phenotypes should be interpreted with caution, since some of the phenotypes could be simply the result of ubiquitin limitation.

Mol Biol Cell, 2005 Jan, 16(1), 372 - 84 Epub 2004 Nov 03.
Actin and septin ultrastructures at the budding yeast cell cortex; Rodal AA et al.; Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling . However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex . To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution . The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments . The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex . Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly . Based on these results, we propose a model for actin function during endocytosis . In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes . These images provide the first high-resolution views of septin organization in cells.

Dev Cell, 2004 Nov, 7(5), 630 - 2
Mitochondrial programmed cell death pathways in yeast; Hardwick JM et al.; Whether or not yeast cell death is altruistic, apoptotic, or otherwise analogous to programmed cell death in mammals is controversial . However, growing attention to cell death mechanisms in yeast has produced several new papers that make a case for ancient origins of programmed death involving mitochondrial pathways conserved between yeast and mammals.

Mol Cell, 2004 Nov 5, 16(3), 487 - 96
A robust toolkit for functional profiling of the yeast genome; Pan X et al.; Study of mutant phenotypes is a fundamental method for understanding gene function . The construction of a near-complete collection of yeast knockouts (YKO) and the unique molecular barcodes (or TAGs) that identify each strain has enabled quantitative functional profiling of Saccharomyces cerevisiae . By using these TAGs and the SGA reporter, MFA1pr-HIS3, which facilitates conversion of heterozygous diploid YKO strains into haploid mutants, we have developed a set of highly efficient microarray-based techniques, collectively referred as dSLAM (diploid-based synthetic lethality analysis on microarrays), to probe genome-wide gene-chemical and gene-gene interactions . Direct comparison revealed that these techniques are more robust than existing methods in functional profiling of the yeast genome . Widespread application of these tools will elucidate a comprehensive yeast genetic network.

Acta Pharmacol Sin, 2004 Nov, 25(11), 1471 - 8
Yeast two-hybrid screening for proteins that interact with alpha1-adrenergic receptors; Zhang T et al.; AIM: To find novel proteins that may bind to alpha1A-adrenergic receptor (alpha1A-AR) and investigate their interactions with the other two alpha1-AR subtypes (alpha1B-AR and alpha1D-AR) with an expectation to provide new leads for the function study of the receptors . METHODS: Yeast two-hybrid assay was performed to screen a human brain cDNA library using the C terminus of alpha1A-AR (alpha1A-AR-CT) as bait . X-Gal assay and o-nitrophenyl-beta-D-galactopyranoside (ONPG) assay were subsequently conducted to further qualitatively or quantitatively confirm the interactions between receptors and the three identified proteins . RESULTS: (1) Selection medium screening identified segments of bone morphogenetic protein-1 (BMP-1), active Bcr-related protein (Abr), and filamin-C as binding partners of alpha1A-AR-CT in yeast cells respectively . Besides, protein segments of BMP-1 and Abr could only specifically interact with alpha1A-AR-CT while filamin-C segment interacted with all three alpha1-AR subtypes . (2) In X-Gal assay, the co-transformants of alpha1A-AR-CT and BMP-1 segments turned strong blue at about 30 min while other positive transformants only developed weak blue at about 5-6 h . (3) In ONPG assay, interaction (shown in beta-galactosidase activity) between alpha1A-AR-CT and BMP-1 segments was about 30 times stronger than that of control (P<0.01), while other positive interactions were only about 2-5 times as strong as those of controls (P<0.05) . CONCLUSION: In yeast cells BMP-1, Abr and/or filamin-C could interact with three alpha1-AR subtypes, among which, interaction between BMP-1 and alpha1A-AR was the strongest while other interactions between proteins and receptors were relatively weak.

Am J Physiol Renal Physiol, 2004 Dec, 287(6), F1102 - 10
MAP kinases and the adaptive response to hypertonicity: functional preservation from yeast to mammals; Sheikh-Hamad D et al.; The adaptation to hypertonicity in mammalian cells is driven by multiple signaling pathways that include p38 kinase, Fyn, the catalytic subunit of PKA, ATM, and JNK2 . In addition to the well-characterized tonicity enhancer (TonE)-TonE binding protein interaction, other transcription factors (and their respective cis elements) can potentially respond to hypertonicity . This review summarizes the current knowledge about the signaling pathways that regulate the adaptive response to osmotic stress and discusses new insights from yeast that could be relevant to the osmostress response in mammals.

Bioelectrochemistry, 2004 Dec, 65(1), 59 - 68
Strong static magnetic field effects on yeast proliferation and distribution; Iwasaka M et al.; The present study focuses on the effects of gradient magnetic fields on the behavior of yeast, such as its proliferation and mass distribution, and evaluates the effects of magnetism on materials in the yeast culture system . Yeast, Saccharomyces cerevisiae, was incubated in a liquid medium under magnetic fields (flux density B = 14 T) . When yeast in a tube was exposed to 9-14 T magnetic fields with a maximum flux density gradient of dB/dx = 94 T/m, where x is the space coordinate, the rate of yeast proliferation under the magnetic fields decreased after 16 h of incubation compared to that of the control group . The physical properties of the yeast culture system were investigated to discover the mechanism responsible for the observed deceleration in yeast proliferation under magnetic fields . Gas pressure inside the yeast culture flask was compared with and without exposure to a magnetic field . The results suggested that the gas pressure inside a flask with 6 T, 60 T/m slowly increased in comparison to the pressure inside a control tube . Due to the diamagnetism of water (medium solution) and yeast, the liquid surface distinctly inclined under gradient magnetic fields, and the hydrostatic force in suspension was strengthened by the diamagnetic forces . In addition, magnetophoresis of the yeast cells in the medium solution exhibited localization of the yeast sedimentation pattern . The roles of magnetically changed gas-transport processes, hydrostatic pressures acting on the yeast, and changes in the distribution of the yeast sedimentation, as well as the possible effects of magnetic fields on yeast respiratory systems in the observed disturbance of the proliferation are discussed.

Aquat Toxicol, 2004 Nov 18, 70(2), 111 - 21
cDNA-directed expression of a functional zebrafish CYP1A in yeast; Chung WG et al.; A cytochrome P450 1A (CYP1A) cDNA was isolated from an adult zebrafish (Danio rerio) library . The 2580-bp clone (GenBank Accession No . AF210727) contained a 62-bp 5'-unstranslated region (UTR), 1557-bp coding region and 962-bp 3'-UTR . The deduced 519-residue protein (calculated molecular weight 58,556, pI = 7.58) shared 74% identity with rainbow trout CYP1A and 57 and 54% identities with mouse and human CYP1A1s, respectively . The zebrafish CYP1A protein coding region was cloned into the pDONR201 entry vector and then transferred to a yeast expression vector pYES-DEST52 . Expression of zebrafish CYP1A in Saccharomyces cerevisiae transformants was induced by galactose to a maximum level of 493 pmol CYP1A per mg microsomal protein or about 8 nmol/l of culture . Recombinant CYP1A protein expressed in yeast was mainly in the denatured P420 form under normal microsomal preparation conditions but when the oxygen concentration was reduced in the buffer by degassing and the yeast cells were maintained at less than 10 degrees C, the integrity of the CYP1A was preserved and it exhibited a characteristic reduced CO-difference spectrum maximum at 448 nm . The recombinant zebrafish CYP1A demonstrated 7-ethoxyresorufin O-deethylase (EROD) activity with an apparent Km (Km(app)) and Vmax values at 30 degrees C of 0.31 +/- 0.04 microM and 0.70 +/- 0.10 nmol/min/nmol CYP, respectively . The recombinant protein also metabolized benzo(a)pyrene with a Km(app) and Vmax values of 5.34 +/- 0.58 microM and 1.16 +/- 0.13 nmol/min/nmol CYP, respectively . These results show the recombinant expression of a functional zebrafish CYP in yeast and validated yeast as a host for heterologous expression of zebrafish CYP1A and potentially for other zebrafish CYPs.

J Inorg Biochem, 2004 Nov, 98(11), 1837 - 50
Inhibition of yeast growth by molybdenum-hydroxylamido complexes correlates with their presence in media at differing pH values; Crans DC et al.; The effects of Mo-hydroxylamido complexes on cell growth were determined in Saccharomyces cerevisiae to investigate the biological effects of four different Mo complexes as a function of pH . Studies with yeast, an eukaryotic cell, are particularly suited to examine growth at different pH values because this organism grows well from pH 3 to 6.5 . Studies can therefore be performed both in the presence of intact complexes and when the complexes have hydrolyzed to ligand and free metal ion . One of the complexes we examined was structurally characterized by X-ray crystallography . Yeast growth was inhibited in media solutions containing added Mo-dialkylhydroxylamido complexes at pH 3-7 . When combining the yeast growth studies with a systematic study of the Mo-hydroxylamido complexes' stability as a function of pH and an examination of their speciation in yeast media, the effects of intact complexes can be distinguished from that of ligand and metal . This is possible because different effects are observed with complex present than when ligand or metal alone is present . At pH 3, the growth inhibition is attributed to the forms of molybdate ion that exist in solution because most of the complexes have hydrolyzed to oxomolybdate and ligand . The monoalkylhydroxylamine ligand inhibited yeast growth at pH 5, 6 and 7, while the dialkylhydroxylamine ligands had little effect on yeast growth . Growth inhibition of the Mo-dialkylhydroxylamido complexes is observed when a complex exists in the media . A complex that is inert to ligand exchange is not effective even at pH 3 where other Mo-hydroxylamido complexes show growth inhibition as molybdate . These results show that the formation of some Mo complexes can protect yeast from the growth inhibition observed when either the ligand or Mo salt alone are present.

Biochem Biophys Res Commun, 2004 Dec 3, 325(1), 183 - 90
Mutational analysis of the interaction between insulin receptor and IGF-I receptor with c-Crk and Crk-L in a yeast two-hybrid system; Klammt J et al.; The SH2/SH3 adapter proteins of the Crk family are potent signal transducers after receptor tyrosine kinase stimulation with insulin or IGF-1 . We have employed a yeast two-hybrid approach and mutational analysis to dissect the capabilities of the insulin receptor and the IGF-I receptor to directly associate with Crk isoforms . Insulin receptor stably recruits full length Crk by association with its SH2 domain in an auto-phosphorylation dependent manner . In contrast, interaction of the IGF-I receptor with the Crk-IISH2 domain was only detectable when Crk-II was truncated in its C-terminal part, indicating the transient nature of this interaction . From these data it can be concluded that members of the insulin receptor family activate Crk proteins in a differential manner.

Br J Nutr, 2004 Oct, 92(4), 557 - 73
The use of high-selenium yeast to raise selenium status: how does it measure up?
Rayman MP.
Selenium-enriched yeast (Se-yeast) is a common form of Se used to supplement the dietary intake of this important trace mineral . However, its availability within the European Union is under threat, owing to concerns expressed by the European Community (EC) Scientific Committee on Food that Se-yeast supplements are poorly characterised and could potentially cause the build up of Se in tissues to toxic levels . The present review examines the validity of these concerns . Diagrams of the biosynthesis and metabolism of Se compounds show which species can be expected to occur in Se-yeast preparations . Se-yeast manufacture is described together with quality-control measures applied by reputable manufacturers . The way in which speciation of Se-yeast is achieved is explained and results on amounts of Se species in various commercial products are tabulated . In all cases described, selenomethionine is the largest single species, accounting for 54-74 % of total Se . Se-yeast is capable of increasing the activity of the selenoenzymes and its bioavailability has been found to be higher than that of inorganic Se sources in all but one study . Intervention studies with Se-yeast have shown the benefit of this form in cancer prevention, on the immune response and on HIV infection . Of about one dozen supplementation studies, none has shown evidence of toxicity even up to an intake level of 800 microg Se/d over a period of years . It is concluded that Se-yeast from reputable manufacturers is adequately characterised, of reproducible quality, and that there is no evidence of toxicity even at levels far above the EC tolerable upper intake level of 300 microg/d.

Traffic, 2004 Dec, 5(12), 1017 - 30
Ncr1p, the yeast ortholog of mammalian niemann Pick C1 protein, is dispensable for endocytic transport; Zhang S et al.; The Niemann Pick C1 protein localizes to late endosomes and plays a key role in the intracellular transport of cholesterol in mammalian cells . Cholesterol and other lipids accumulate in a lysosomal or late endosomal compartment in cells lacking normal NPC1 function . Other than accumulation of lipids, defects in lysosomal retroendocytosis, sorting of a multifunctional receptor and endosomal movement have also been detected in NPC1 mutant cells . Ncr1p is an ortholog of NPC1 in the budding yeast Saccharomyces cerevisiae . In this study, we show that Ncr1p is a vacuolar membrane protein that transits through the biosynthetic vacuolar protein sorting pathway, and that it can be solubilized by Triton X-100 at 4 degrees C . Using well-established assays, we demonstrate that the absence of Ncr1p had no effect on fluid phase and receptor- mediated endocytosis, biosynthetic delivery to the vacuole, retrograde transport from endosome to Golgi and ubiquitin- and nonubiquitin-dependent multivesicular body sorting . We conclude that Ncr1p does not have an essential role in known endocytic transport pathways in yeast.

Biochemistry (Mosc), 2004 Sep, 69(9), 1044 - 50
Positioning of a nucleosome on mouse satellite DNA inserted into a yeast plasmid is determined by its DNA sequence and an adjacent nucleosome; Kiryanov GI et al.; It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence . To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene . We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro . The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well . Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter . Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.

Biochemistry (Mosc), 2004 Sep, 69(9), 1025 - 33
Calcium-dependent nonspecific permeability of the inner mitochondrial membrane is not induced in mitochondria of the yeast Endomyces magnusii; Deryabina YI et al.; Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore) . For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria . Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction . E . magnusii mitochondria were highly resistant to the above-mentioned substances . Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters . Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured . It is thus evident that E . magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.

Mikrobiologiia, 2004 Jul-Aug, 73(4), 558 - 66
{Community structure of yeast fungi in forest biogeocenoses}
{Correlation between the cellular content of mobile water and the viability of lyophilized yeast cells}
{No authors listed}

Spin-echo NMR studies showed that lyophilized yeast cells contain isolated mobile water (IMW), whose content varied from 0.25% (of the dry weight of cells) in the lyophilized exponential-phase yeast cells to 3.8% in the lyophilized lag-phase and stationary-phase yeast cells . The viability rate of yeast cells varied from 20% in the lyophilized preparation of exponential-phase cells to 86% in the lyophilized preparation of early-stationary-phase cells . In the lyophilized preparation of yeast cells grown in a chemostat mode at a constant specific rate, the content of IMW depended on the growth-limiting factor, being minimal in the case of growth limitation by carbon source . In the latter case, the viability rate of cells was also minimal . The data obtained show that there is a correlation between the IMW content and the viability rate of yeast cells in lyophilized preparations.

Nucleic Acids Res, 2004 Nov 01, 32(19), 5894 - 906 Print 2004.
Domain organization of the yeast histone chaperone FACT: the conserved N-terminal domain of FACT subunit Spt16 mediates recovery from replication stress; O'Donnell AF et al.; The abundant nuclear complex termed FACT affects several DNA transactions in a chromatin context, including transcription, replication, and repair . Earlier studies of yeast FACT, which indicated the apparent dispensability of conserved sequences at the N terminus of the FACT subunit Cdc68/Spt16, prompted genetic and biochemical studies reported here that suggest the domain organization for Spt16 and the other FACT subunit Pob3, the yeast homolog of the metazoan SSRP1 protein . Our findings suggest that each FACT subunit is a multidomain protein, and that FACT integrity depends on Pob3 interactions with the Spt16 Mid domain . The conserved Spt16 N-terminal domain (NTD) is shown to be without essential function during normal growth, but becomes important under conditions of replication stress . Genetic interactions suggest that some functions carried out by the Spt16 NTD may be partially redundant within FACT.

Genome Res, 2004 Nov, 14(11), 2319 - 29
Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using transformation-associated recombination in yeast; Becker M et al.; Trypanosoma brucei switches between variant surface glycoproteins (VSGs) allowing immune escape . The active VSG is in one of many telomeric bloodstream form VSG expression sites (BESs), also containing expression site-associated genes (ESAGs) involved in host adaptation . The role of BES sequence diversity in parasite virulence can best be understood through analysis of the full repertoire of BESs from a given T . brucei strain . However, few BESs have been cloned, as telomeres are highly underrepresented in standard libraries . We devised a strategy for isolating the repertoire of T . brucei 427 BES-containing telomeres in Saccaromyces cerevisiae by using transformation-associated recombination (TAR) . We isolated 182 T . brucei 427 BES TAR clones, 167 of which could be subdivided into minimally 17 BES groups . This set gives us the first view of the breadth and diversity of BESs from one T . brucei strain . Most BESs ranged between 40 and 70 kb (average, 57 +/- 17 kb) and contained most identified ESAGs . Phylogenetic comparison of the cohort of BES promoter and ESAG6 sequences did not show similar trees, indicating rapid evolution most likely mediated by sequence exchange between BESs . This cloning strategy could be used for any T . brucei strain, facilitating research on the biodiversity of telomeric gene families and host-pathogen interactions.

Genes Dev, 2004 Nov 15, 18(22), 2785 - 97 Epub 2004 Nov 01.
Mitochondrial fission proteins regulate programmed cell death in yeast; Fannjiang Y et al.; The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery . However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells . In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli . Two Dnm1-interacting factors also regulate yeast cell death . The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission . In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast . Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL . Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

J Cell Biol, 2004 Nov 8, 167(3), 433 - 43 Epub 2004 Nov 01.
A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast; Alberghina L et al.; Saccharomyces cerevisiae must reach a carbon source-modulated critical cell size, protein content per cell at the onset of DNA replication (Ps), in order to enter S phase . Cells grown in glucose are larger than cells grown in ethanol . Here, we show that an increased level of the cyclin-dependent inhibitor Far1 increases cell size, whereas far1 Delta cells start bud emergence and DNA replication at a smaller size than wild type . Cln3 Delta, far1 Delta, and strains overexpressing Far1 do not delay budding during an ethanol glucose shift-up as wild type does . Together, these findings indicate that Cln3 has to overcome Far1 to trigger Cln-Cdc28 activation, which then turns on SBF- and MBF-dependent transcription . We show that a second threshold is required together with the Cln3/Far1 threshold for carbon source modulation of Ps . A new molecular network accounting for the setting of Ps is proposed.

Chemosphere, 2004 Dec, 57(11), 1649 - 55
Determination of estrogenic activity by LYES-assay (yeast estrogen screen-assay assisted by enzymatic digestion with lyticase); Schultis T et al.; In order to enhance the sensitivity and the speed of the yeast estrogen screen (YES)-assay, which has been established in many laboratories for the determination of estrogenic activity of compounds and environmental samples, the LYES-assay, a modified version of the YES-assay including a digestion step with the enzyme lyticase, was developed . With the LYES-assay the estrogenic activities of natural (17beta-estradiol E2 and estrone), synthetic (17alpha-ethinylestradiol EE2) and pharmaceutical estrogens (diethylstilbestrol DES) as well as xenoestrogens (4-nonylphenol NP and five parabens) were determined and compared with the results obtained by other in vitro-assays namely the conventional YES-assay, the E-Screen-assay (MCF-7 breast tumor cell proliferation) and a receptor binding-assay (RB) with human estrogen receptors hER-alpha and hER-beta . In the case of E2 the LYES-assay had a significantly lower limit of quantification (LOQ) than the conventional YES-assay and even two orders of magnitude lower than the RB-assay . Compared to the E-Screen-assay the LOQ of the LYES-assay was almost one order of magnitude higher . The time required to perform the LYES-assay was as little as seven hours compared to three to five days for the conventional YES-assay . Thus, the LYES-assay is a very good alternative to existing estrogenic in vitro-assays, since it has a good sensitivity, is cheap and much faster than the other assays.

Analyst, 2004 Oct, 129(10), 866 - 9
Using yeast to shed light on DNA damaging toxins and irradiation; Knight AW; This unlikely tale of research and development takes us from a Pacific jellyfish to fluorescent green yeast in space, via the high tech world of pharmaceutical screening and an encounter with the YETI . It charts the history of academic research project that through multidisciplinary collaboration and innovation has led to a commercial venture with applications in a number of diverse fields.

Huan Jing Ke Xue, 2004 Jul, 25(4), 155 - 8
{Modification and application of recombinant yeast bioassay for measuring the AhR ligand activity}; Wang W et al.; A recombinant yeast bioassay, a yeast Saccharomyces cerevisiae, in which the human AhR and ARNT complex are coexpressed, is one of the methods to screen the active AhR agonist . In this study, the original agonist test was modified . The exposure time was reduced from 18 to 8 hours when experiment was under the following conditions: (1) the yeast was cultured in 0.2% glucose medium for 24 hours; (2) chemical exposure was carried in 2% galactose medium in glass tube . Finally, the AhR acitivity of hexachlorobenzene and pentachlorobenzene were assessed, and their toxicity equivalent factors were found to be 0.018629 and 0.000294, respectively.

Yeast, 2004 Oct 30, 21(14), 1219 - 32
The yeast rRNA biosynthesis factor Ebp2p is also required for efficient nuclear division; Ionescu CN et al.; Molecular genetic analysis of the yeast Ebp2 protein has revealed that it is an essential, nucleolar protein that functions in the rRNA biosynthesis pathway . Temperature-sensitive ebp2-1 mutants are defective in the processing of the 27 SA precursor rRNA, and the point substitutions that disrupt this activity cluster towards the central, more highly conserved region of the Ebp2 protein . We report here that other ebp2 mutants exhibit deficiencies associated with defects in chromosome segregation . Yeast cells bearing a 50 amino acid C-terminal truncation allele (ebp2 delta C50) display a slow-growth phenotype and exhibit an increased percentage of cells with the nucleus positioned at the bud neck . The ebp2-1 and ebp2 delta C50 alleles genetically complement each other, and ebp2 delta C50 mutants exhibit nuclear division defects that are distinct from the rRNA biosynthesis-related phenotypes of ebp2-1 mutants . Cytological and FACS analysis of the ebp2 delta C50 deletion mutants indicate that the chromosome segregation related activities of the Ebp2 protein are monitored by Mad2p, a mitotic checkpoint protein . The finding that yeast Ebp2p functions in nuclear division is consistent with the growing body of evidence that supports the role that human EBP2 plays in chromosome segregation . copyright 2004 John Wiley & Sons, Ltd.

Genetics, 2004 Oct, 168(2), 775 - 83
Meiotic chromosome synapsis in yeast can occur without spo11-induced DNA double-strand breaks; Bhuiyan H et al.; Proper chromosome segregation and formation of viable gametes depend on synapsis and recombination between homologous chromosomes during meiosis . Previous reports have shown that the synaptic structures, the synaptonemal complexes (SCs), do not occur in yeast cells with the SPO11 gene removed . The Spo11 enzyme makes double-strand breaks (DSBs) in the DNA and thereby initiates recombination . The view has thus developed that synapsis in yeast strictly depends on the initiation of recombination . Synapsis in some other species (Drosophila melanogaster and Caenorhabditis elegans) is independent of recombination events, and SCs are found in spo11 mutants . This difference between species led us to reexamine spo11 deletion mutants of yeast . Using antibodies against Zip1, a SC component, we found that a small fraction (1%) of the spo11 null mutant cells can indeed form wild-type-like SCs . We further looked for synapsis in a spo11 mutant strain that accumulates pachytene cells (spo11Delta ndt80Delta), and found that the frequency of cells with apparently complete SC formation was 10% . Other phenotypic criteria, such as spore viability and homologous chromosome juxtaposition measured by FISH labeling of chromosomal markers, agree with several previous reports of the spo11 mutant . Our results demonstrate that although the Spo11-induced DSBs obviously promote synapsis in yeast, the presence of Spo11 is not an absolute requirement for synapsis.

Genetics, 2004 Oct, 168(2), 759 - 74
A functional analysis reveals dependence on the anaphase-promoting complex for prolonged life span in yeast; Harkness TA et al.; Defects in anaphase-promoting complex (APC) activity, which regulates mitotic progression and chromatin assembly, results in genomic instability, a hallmark of premature aging and cancer . We investigated whether APC-dependent genomic stability affects aging and life span in yeast . Utilizing replicative and chronological aging assays, the APC was shown to promote longevity . Multicopy expression of genes encoding Snf1p (MIG1) and PKA (PDE2) aging-pathway components suppressed apc5CA phenotypes, suggesting their involvement in APC-dependent longevity . While it is known that PKA inhibits APC activity and reduces life span, a link between the Snf1p-inhibited Mig1p transcriptional modulator and the APC is novel . Our mutant analysis supports a model in which Snf1p promotes extended life span by inhibiting the negative influence of Mig1p on the APC . Consistent with this, we found that increased MIG1 expression reduced replicative life span, whereas mig1Delta mutations suppressed the apc5CA chronological aging defect . Furthermore, Mig1p and Mig2p activate APC gene transcription, particularly on glycerol, and mig2Delta, but not mig1Delta, confers a prolonged replicative life span in both APC5 and acp5CA cells . However, glucose repression of APC genes was Mig1p and Mig2p independent, indicating the presence of an uncharacterized factor . Therefore, we propose that APC-dependent genomic stability is linked to prolonged longevity by the antagonistic regulation of the PKA and Snf1p pathways.

Genetics, 2004 Oct, 168(2), 733 - 46
Lrg1p Is a Rho1 GTPase-activating protein required for efficient cell fusion in yeast; Fitch PG et al.; To identify additional cell fusion genes in Saccharomyces cerevisiae, we performed a high-copy suppressor screen of fus2Delta . Higher dosage of three genes, BEM1, LRG1, and FUS1, partially suppressed the fus2Delta cell fusion defect . BEM1 and FUS1 were high-copy suppressors of many cell-fusion-defective mutations, whereas LRG1 suppressed only fus2Delta and rvs161Delta . Lrg1p contains a Rho-GAP homologous region . Complete deletion of LRG1, as well as deletion of the Rho-GAP coding region, caused decreased rates of cell fusion and diploid formation comparable to that of fus2Delta . Furthermore, lrg1Delta caused a more severe mating defect in combination with other cell fusion mutations . Consistent with an involvement in cell fusion, Lrg1p localized to the tip of the mating projection . Lrg1p-GAP domain strongly and specifically stimulated the GTPase activity of Rho1p, a regulator of beta(1-3)-glucan synthase in vitro . beta(1-3)-glucan deposition was increased in lrg1Delta strains and mislocalized to the tip of the mating projection in fus2Delta strains . High-copy LRG1 suppressed the mislocalization of beta(1-3) glucan in fus2Delta strains . We conclude that Lrg1p is a Rho1p-GAP involved in cell fusion and speculate that it acts to locally inhibit cell wall synthesis to aid in the close apposition of the plasma membranes of mating cells.

Bioinformatics . 2004 Oct 28; {Epub ahead of print}
Predicting protein localization in budding yeast; Chou KC et al.; MOTIVATION: Most of the existing methods in predicting protein subcellular location were used to deal with the cases limited within the scope from 2 to 5 localizations, and only a few of them can be effectively extended to cover the cases of 12-14 localizations . This is because the more the locations involved, the poorer the success rate would be . Besides, some proteins may occur in several different subcellular locations, i.e., bear the feature of "multiplex locations" . So far there is no method that can be used to effectively treat the difficult multiplex location problem . The present study was initiated in an attempt to address (1) how to efficiently identify the localization of a query protein among many possible subcellular locations, and (2) how to deal with the case of multiplex locations . RESULTS: By hybridizing gene ontology, functional domain, and pseudo amino acid composition approaches, a new method has been developed that can be used to predict subcellular localization of proteins with multiplex location feature . A global analysis of the proteins in budding yeast classified into 22 locations was performed by jackknife cross-validation with the new method . The overall success identification rate thus obtained is 70% . In contrast to this, the corresponding rates obtained by some other existing methods were only 13-14%, indicating that the new method is very powerful and promissing . Furthermore, predictions were made for the 4 proteins whose localizations could not be determined by experiments, as well as for the 236 proteins whose localizations in budding yeast were ambiguous according to experimental observations . However, according to our predicted results, many of these "ambiguous proteins" were found to have a same score and ranking for several different subcellular locations, implying that they may simultaneously exist, or move around, in these locations . This finding is intriguing because it reflects the dynamic feature of these proteins in a cell that may be associated with some special biological functions.

Mol Cell Biol, 2004 Nov, 24(22), 10026 - 35
Global chromatin structure of 45,000 base pairs of chromosome III in a- and alpha-cell yeast and during mating-type switching; Ercan S et al.; Directionality of yeast mating-type switching has been attributed to differences in chromatin structure for the left arm of chromosome III . We have mapped the structure of approximately 45 kbp of the left arm of chromosome III in a and alpha cells in logarithmically growing cultures and in a cells during switching . Distinctive features of chromatin structure were the occurrence of DNase I-hypersensitive sites in the promoter region of nearly every gene and some replication origins and the presence of extended regions of positioned nucleosomes in approximately 25% of the open reading frames . Other than the recombination enhancer, chromatin structures were identical in the two cell types . Changes in chromatin structure during switching were confined to the recombination enhancer . This unbiased analysis of an extended region of chromatin reveals that significant features of organized chromatin exist for the entire region, and these features are largely static with respect to mating type and mating-type switching . Our analysis also shows that primary chromatin structure does not cause the documented differences in recombinational frequency of the left arm of chromosome III in yeast a and alpha cells.

Mol Cell Biol, 2004 Nov, 24(22), 9873 - 86
Novel functional dissection of the localization-specific roles of budding yeast polo kinase Cdc5p; Park JE et al.; Budding yeast polo kinase Cdc5p localizes to the spindle pole body (SPB) and to the bud-neck and plays multiple roles during M-phase progression . To dissect localization-specific mitotic functions of Cdc5p, we tethered a localization-defective N-terminal kinase domain of Cdc5p (Cdc5pDeltaC) to the SPB or to the bud-neck with components specifically localizing to one of these sites and characterized these mutants in a cdc5Delta background . Characterization of a viable, SPB-localizing, CDC5DeltaC-CNM67 mutant revealed that it is defective in timely degradation of Swe1p, a negative regulator of Cdc28p . Loss of BFA1, a negative regulator of mitotic exit, rescued the lethality of a neck-localizing CDC5DeltaC-CDC12 or CDC5DeltaC-CDC3 mutant but yielded severe defects in cytokinesis . These data suggest that the SPB-associated Cdc5p activity is critical for both mitotic exit and cytokinesis, whereas the bud neck-localized Cdc5p is required for proper Swe1p regulation . Interestingly, a cdc5Delta bfa1Delta swe1Delta triple mutant is viable but grows slowly, whereas cdc5Delta cells bearing both CDC5DeltaC-CNM67 and CDC5DeltaC-CDC12 grow well with only a mild cell cycle delay . Thus, SPB- and the bud-neck-localized Cdc5p control most of the critical Cdc5p functions and downregulation of Bfa1p and Swe1p at the respective locations are two critical factors that require Cdc5p.

Mol Cell Biol, 2004 Nov, 24(22), 9771 - 85
Amino acid signaling in yeast: casein kinase I and the Ssy5 endoprotease are key determinants of endoproteolytic activation of the membrane-bound Stp1 transcription factor; Abdel-Sater F et al.; Saccharomyces cerevisiae cells possess a plasma membrane sensor able to detect the presence of extracellular amino acids and then to activate a signaling pathway leading to transcriptional induction of multiple genes, e.g., AGP1, encoding an amino acid permease . This sensing function requires the permease-like Ssy1 and associated Ptr3 and Ssy5 proteins, all essential to activation, by endoproteolytic processing, of the membrane-bound Stp1 transcription factor . The SCF(Grr1) ubiquitin-ligase complex is also essential to AGP1 induction, but its exact role in the amino acid signaling pathway remains unclear . Here we show that Stp1 undergoes casein kinase I-dependent phosphorylation . In the yck mutant lacking this kinase, Stp1 is not cleaved and AGP1 is not induced in response to amino acids . Furthermore, we provide evidence that Ssy5 is the endoprotease responsible for Stp1 processing . Ssy5 is significantly similar to serine proteases, its self-processing is a prerequisite for Stp1 cleavage, and its overexpression causes inducer-independent Stp1 cleavage and high-level AGP1 transcription . We further show that Stp1 processing also requires the SCF(Grr1) complex but is insensitive to proteasome inhibition . However, Stp1 processing does not require SCF(Grr1), Ssy1, or Ptr3 when Ssy5 is overproduced . Finally, we describe the properties of a particular ptr3 mutant that suggest that Ptr3 acts with Ssy1 in amino acid detection and signal initiation . We propose that Ssy1 and Ptr3 form the core components of the amino acid sensor . Upon detection of external amino acids, Ssy1-Ptr3 likely allows-in a manner dependent on SCF(Grr1)-the Ssy5 endoprotease to gain access to and to cleave Stp1, this requiring prior phosphorylation of Stp1 by casein kinase I.

Sci STKE . 2004 Oct 26;2004(256):re17.
Selective gene expression in multigene families from yeast to mammals; Dalgaard JZ et al.; Exclusive gene expression, where only one member of a gene or gene cassette family is selected for expression, plays an important role in the establishment of cell identity in several biological systems . Here, we compare four such systems: mating-type switching in fission and budding yeast, where cells choose between expressing one of the two different mating-type cassettes, and immunoglobulin and odorant receptor gene expression in mammals, where the number of gene choices is substantially higher . The underlying mechanisms that establish this selective expression pattern in each system differ in almost every detail . In all four systems, once a successful gene activation event has taken place, a feedback mechanism affects the fate of the cell . In the mammalian systems, feedback is mediated by the expressed cell surface receptor to ensure monoallelic gene expression, whereas in the yeasts, the expressed gene cassette at the mating-type locus affects donor choice during the subsequent switching event.

J Biol Chem, 2005 Jan 21, 280(3), 1854 - 63 Epub 2004 Oct 26.
A Role for Pet100p in the Assembly of Yeast Cytochrome c Oxidase: INTERACTION WITH A SUBASSEMBLY THAT ACCUMULATES IN A pet100 MUTANT; Church C et al.; The biogenesis of multimeric protein complexes of the inner mitochondrial membrane in yeast requires a number of nuclear-coded ancillary proteins . One of these, Pet100p, is required for cytochrome c oxidase . Previous studies have shown that Pet100p is not required for the synthesis, processing, or targeting of cytochrome c oxidase subunits to the mitochondrion nor for heme A biosynthesis . Here, we report that Pet100p does not affect the localization of cytochrome c oxidase subunit polypeptides to the inner mitochondrial membrane but instead functions after they have arrived at the inner membrane . We have also localized Pet100p to the inner mitochondrial membrane in wild type cells, where it is present in a subassembly (Complex A) with cytochrome c oxidase subunits VII, VIIa, and VIII . Pet100p does not interact with the same subunits after they have been assembled into the holoenzyme . In addition, we have identified two subassemblies that are present in pet100 null mutant cells: one subassembly (Complex A') is composed of subunits VII, VIIa, and VIII but not Pet100p, and another subassembly (Complex B) is composed of subunits Va and VI . Because pet100 null mutant cells lack assembled cytochrome c oxidase but accumulate Complexes A' and B it appears likely that these subassemblies of cytochrome c oxidase subunits are intermediates along an assembly pathway for holocytochrome c oxidase and that Pet100p functions in this pathway to facilitate the interaction(s) between Complex A' and other cytochrome c oxidase subassemblies and subunits.

J Theor Biol, 2004 Dec 7, 231(3), 443 - 58
Modeling the synchronization of yeast respiratory oscillations; Henson MA; The budding yeast Saccharomyces cerevisiae exhibits autonomous oscillations when grown aerobically in continuous culture with ethanol as the primary carbon source . A single cell model that includes the sulfate assimilation and ethanol degradation pathways recently has been developed to study these respiratory oscillations . We utilize an extended version of this single cell model to construct large cell ensembles for investigation of a proposed synchronization mechanism involving hydrogen sulfide . Ensembles with as many as 10,000 cells are used to simulate population synchronization and to compute transient number distributions from asynchronous initial cell states . Random perturbations in intracellular kinetic parameters are introduced to study the synchronization of single cells with small variations in their unsynchronized oscillation periods . The cell population model is shown to be consistent with available experimental data and to provide insights into the regulatory mechanisms responsible for the synchronization of yeast metabolic oscillations.

Cancer Gene Ther, 2004 Dec, 11(12), 790 - 6
A novel fusion suicide gene yeast CDglyTK plays a role in radio-gene therapy of nasopharyngeal carcinoma; Xia K et al.; To investigate a novel suicide gene for nasopharyngeal carcinoma (NPC) therapy, the yCDglyTK gene was constructed by fusing yeast cytosine deaminase (CD) and herpes simplex type 1 thymidine kinase . The expression of the yCDglyTK gene was detected by RT-PCR and Western blotting, and its bioactivity was demonstrated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay . An animal study was carried out in which BALB/C nude mice bearing yCDglyTK gene-modified tumors were treated with prodrugs and radiation . Our results revealed that the yCDglyTK gene could be expressed in CNE-2 cells in vitro . In MTT analysis, at the transfection rate of 10%, 66% cells were killed . The synergistic effect of CD and TK showed 91% of yCDglyTK-transfected cells were killed with the treatment of 5-fluorocytosine (5-FC) alone, 60% killed with ganciclovir (GCV) alone, and 75% killed with 5-FC and GCV together . In vivo, the tumor volume in all of the four prodrugs and/or radiation-treated groups were significantly different from that in the PBS-controlled group (P<.01); also yCDglyTK+prodrug+radiation group was different from the other three groups (P<.05) . Our findings suggested there was a synergistic antitumor effect when combining suicide gene therapy and radiation, and yCDglyTK has potent antitumor efficacy and may be a candidate suicide gene for cancer therapy.

J Biol Chem, 2005 Jan 21, 280(3), 2331 - 40 Epub 2004 Oct 21.
The Yeast Mitochondrial Citrate Transport Protein: CHARACTERIZATION OF TRANSMEMBRANE DOMAIN III RESIDUE INVOLVEMENT IN SUBSTRATE TRANSLOCATION; Ma C et al.; Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to hydrophilic, cysteine-specific methanethiosulfonate reagents, enabled identification of the water-accessible surface of this domain and suggested its potential participation in the formation of a portion of the substrate translocation pathway . To evaluate this idea, we conducted a detailed characterization of the functional properties of 20 TMDIII single-Cys substitution mutants . Kinetic studies indicate that the A118C, S123C, and K134C mutants displayed a 3- to 7-fold increase in K(m) . Moreover, the A118C mutation caused a doubling of the V(max) value, whereas the S123C, E131C, and K134C mutations caused V(max) to dramatically decrease, resulting in a reduction of the catalytic efficiencies of these three mutants by >97% . Examination of the ability of citrate to protect against the inhibition mediated by sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) indicated that citrate conferred significant protection of cysteines substituted at eight water-accessible locations (i.e . Gly-115, Leu-116, Gly-117, Leu-121, Ser-123, Val-127, Glu-131, and Thr-135), but not at other sites . Importantly, similar levels of protection were observed at both 4 degrees C and 20 degrees C . The temperature independence of the protection indicates that substrate binding and/or occupancy of the transport pathway sterically blocks the access of MTSES to these sites, thereby providing direct protection, without involvement of a major protein conformational change . The significance of these extensive functional investigations is discussed in terms of the three-dimensional CTP homology model that we previously developed and a new model of the dimer interface.

J Theor Biol, 2005 Jan 7, 232(1), 83 - 91
DNA in phosphorus atom representation: the heteronomous double helices of poly(dA).poly(dT) and poly(dG).poly(dC) and simulation of the yeast genome and of a human chromosome DNA; Hanzalek P et al.; We extracted phosphorus atom coordinates from the database of DNA crystal structures and calculated geometrical parameters needed to reproduce the crystal structures in the phosphorus atom representation . Using the geometrical parameters we wrote a piece of software assigning the phosphorus atom coordinates to the DNA of any nucleotide sequence . The software demonstrates non-negligible influence of the primary structure on DNA helicity, which may stand behind the heteromonous double helices of poly(dA).poly(dT) and poly(dG).poly(dC) . In addition, the software is so simple that it makes possible to simulate the "crystal" structures of not only viral DNAs, but also the whole genome of Saccharomyces cerevisiae as well as the DNA human chromosome 22 having dozens of megabases in length.

Mol Biol Evol . 2004 Oct 20; {Epub ahead of print}
An Exponential Core in the Heart of the Yeast Protein Interaction Network; Pereira-Leal JB et al.; Protein interactions in the budding yeast have been shown to form a scale-free network, a feature of other organized networks like bacterial and archaeal metabolism and the world wide web . Here, we study the connections established by yeast proteins and discover a preferential attachment between essential proteins . The essential connections are long-ranged and form a sub-network where the giant component includes 97% of these proteins . Unexpectedly, this sub-network displays an exponential connectivity distribution, in sharp contrast with the scale-free topology of the complete network . Furthermore, the wide phylogenetic extent of these core proteins and interactions provides evidence that they represent the ancestral state of the yeast protein interaction network . Finally, we propose that this core exponential network may represent a generic scaffold around which organism- and taxon-specific proteins and interactions coalesce.

Mol Cell, 2004 Oct 22, 16(2), 199 - 209
Global position and recruitment of HATs and HDACs in the yeast genome; Robert F et al.; Chromatin regulators play fundamental roles in the regulation of gene expression and chromosome maintenance, but the regions of the genome where most of these regulators function has not been established . We explored the genome-wide occupancy of four different chromatin regulators encoded in Saccharomyces cerevisiae . The results reveal that the histone acetyltransferases Gcn5 and Esa1 are both generally recruited to the promoters of active protein-coding genes . In contrast, the histone deacetylases Hst1 and Rpd3 are recruited to specific sets of genes associated with distinct cellular functions . Our results provide new insights into the association of histone acetyltransferases and histone deacetylases with the yeast genome, and together with previous studies, suggest how these chromatin regulators are recruited to specific regions of the genome.

Adv Biophys, 2004, 38, 215 - 32
Molecular mechanism of VDE-initiated intein homing in yeast nuclear genome; Fukuda T et al.; In Saccharomyces cerevisiae, VMA1 intein encodes a homing endonuclease termed VDE which is produced by an autocatalytic protein splicing reaction . VDE introduces a DSB at its recognition sequence on intein-minus allele, resulting in the lateral transfer of VMA1 intein . In this review, we summarize a decade of in vitro study on VDE and describe our recent study on the in vivo behavior of both VDE and host proteins involved in intein mobility . Meiotic DSBs caused by VDE are repaired in the similar pathway to that working in meiotic recombination induced by Spo11p-mediated DSBs . Meiosis-specific DNA cleavage and homing is shown to be guaranteed by the two distinct mechanisms, the subcellular localization of VDE and a requirement of premeiotic DNA replication . Based on these lines of evidence, we present the whole picture of molecular mechanism of VDE-initiated homing in yeast cells.

Mutat Res, 2004 Nov 22, 556(1-2), 201 - 8
Non-homologous end joining dependency of gamma-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells; Heidenreich E et al.; There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions . Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells . During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair . Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis . An artificially imposed excess of DSBs by gamma-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions . By the use of DNA ligase IV-deficient strains we detected that the majority of the gamma-induced adaptive mutations were also dependent on a functional NHEJ pathway . This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs . Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development.

Biochemistry, 2004 Oct 26, 43(42), 13564 - 78
A fluorescent alpha-factor analogue exhibits multiple steps on binding to its G protein coupled receptor in yeast; Bajaj A et al.; The yeast alpha-factor receptor encoded by the STE2 gene is a member of the extended family of G protein coupled receptors (GPCRs) involved in a wide variety of signal transduction pathways . We report here the use of a fluorescent alpha-factor analogue {K(7)(NBD), Nle(12)} alpha-factor (Lys(7) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), norleucine(12) alpha-factor) in conjunction with flow cytometry and fluorescence microscopy to study binding of ligand to the receptor . Internalization of the fluorescent ligand following receptor binding can be monitored by fluorescence microscopy . The use of flow cytometry to detect binding of the fluorescent ligand to intact yeast cells provides a sensitive and reproducible assay that can be conducted at low cell densities and is relatively insensitive to fluorescence of unbound and nonspecifically bound ligand . Using this assay, we determined that some receptor alleles expressed in cells lacking the G protein alpha subunit exhibit a higher equilibrium binding affinity for ligand than the same alleles expressed in isogenic cells containing the normal complement of G protein subunits . On the basis of time-dependent changes in the intensity and shape of the emission spectrum of {K(7)(NBD),Nle(12)} alpha-factor during binding, we infer that the ligand associates with receptors via a two-step process involving an initial interaction that places the fluorophore in a hydrophobic environment, followed by a conversion to a state in which the fluorophore moves to a more polar environment.

Curr Genet . 2004 Oct 13; {Epub ahead of print}
Activity of the yeast Tat2p tryptophan permease is sensitive to the anti-tumor agent 4-phenylbutyrate; Liu M et al.; 4-Phenylbutyrate (PB) induces differentiation and is being intensively studied as a treatment for brain, prostate, breast, and hematopoietic cancer . While many different primary targets for PB have been proposed, the mechanism by which it causes cellular differentiation remains unknown . To identify the primary cellular target, we investigated its effects on Saccharomyces cerevisiae and showed that it inhibits tryptophan transport . We show here that PB and sorbic acid induce an ubiquitin-dependent turnover of the tryptophan permease Tat2p . However, the inhibition of transport is not a consequence of the loss of Tat2p, since it also occurs when turnover is prevented by deleting the Tat2p ubiquitination sites . When we tested the effects of PB and other growth inhibitory agents on the growth of amino acid auxotrophs, we found that several auxotrophs are hypersensitive to a number of chemically unrelated agents, including PB and some, but not all, weak acids; and this sensitivity is due to the inhibition of amino acid transport . For the inhibitory weak acids, inhibition is not confined to aromatic amino acid auxotrophs, nor is it a general weak acid stress response, since the degree of inhibition is independent of weak acid hydrophobicity and p K(a) . Our results show that diverse agents affect the activity of the Tat2p permease rather than its stability and suggest the hypothesis that the anti-neoplastic action of PB is due to a decrease in the activity of surface receptors or other membrane proteins needed to maintain the transformed state.

Chem Commun (Camb), 2004 Oct 21, (20), 2338 - 9 Epub 2004 Sep 07.
A novel strategy for the preparation of arylhydroxylamines: chemoselective reduction of aromatic nitro compounds using bakers' yeast; Li F et al.; Using bakers' yeast as a biocatalyst, the chemoselective reduction of aromatic nitro compounds bearing electron-withdrawing groups gave the corresponding hydroxylamines with good to excellent conversion under mild conditions.

Beijing Da Xue Xue Bao, 2004 Oct, 36(5), 514 - 8
{Identification of proteins interacting with androgen receptor- associated coregulator 267-alpha (ARA267-alpha) with the yeast two-hybrid system.}; Wang X et al.; OBJECTIVE: To search for proteins interacting with ARA267-alpha with the yeast two-hybrid system in order to further investigate the function of ARA267-alpha . METHODS: We screened a pretransformed human brain cDNA library with the pGBKT7-PHD-SET recombinant plasmid as a bait which express four PHD(plant homeodomain) and one SET{Su(var)3-9, Enhancer-of-zeste, Trithorax} conserved domains in ARA267-alpha . The plasmids in positive yeast clones were selectively identified by restriction analysis and DNA sequencing . The interactions were retested by yeast two-hybrid assay . RESULTS: There were about six hundreds positive yeast clones on SD/-Ade/-His/-Leu/-Trp/2.5 mmol/L 3-AT/ X-alpha-Gal high-stringency selection plates . The pACT2-cDNA plasmids in sixty-five yeast clones were isolated and thirty-five cDNA inserts were sequenced . Sixteen different genes, including DR6(death receptor-6), PIAS3 (protein inhibitor of activated STAT3)and RanBPM(Ran-binding protein in the microtubule-organizing center), were identified after BLAST in GenBank . The yeast two-hybrid retest showed that all but RanBPM were true interactors of ARA267-alpha-PHD-SET . CONCLUSION: The ARA267-alpha-PHD-SET can interact with several distinct proteins . This suggests that ARA267-alpha is a protein having multiple functions . RanBPM might be a transcriptional factor.

Genome Res, 2004 Oct, 14(10B), 2093 - 101
A gateway-compatible yeast one-hybrid system; Deplancke B et al.; Since the advent of microarrays, vast amounts of gene expression data have been generated . However, these microarray data fail to reveal the transcription regulatory mechanisms that underlie differential gene expression, because the identity of the responsible transcription factors (TFs) often cannot be directly inferred from such data sets . Regulatory TFs activate or repress transcription of their target genes by binding to cis-regulatory elements that are frequently located in a gene's promoter . To understand the mechanisms underlying differential gene expression, it is necessary to identify physical interactions between regulatory TFs and their target genes . We developed a Gateway-compatible yeast one-hybrid (Y1H) system that enables the rapid, large-scale identification of protein-DNA interactions using both small (i.e., DNA elements of interest) and large (i.e., gene promoters) DNA fragments . We used four well-characterized Caenorhabditis elegans promoters as DNA baits to test the functionality of this Y1H system . We could detect approximately 40% of previously reported TF-promoter interactions . By performing screens using two complementary libraries, we found novel potentially interacting TFs for each promoter . We recapitulated several of the Y1H-based protein-DNA interactions using luciferase reporter assays in mammalian cells . Taken together, the Gateway-compatible Y1H system will allow the high-throughput identification of protein-DNA interactions and may be a valuable tool to decipher transcription regulatory networks.

Genes Dev, 2004 Nov 1, 18(21), 2663 - 75 Epub 2004 Oct 15.
Telomerase- and recombination-independent immortalization of budding yeast; Maringele L et al.; It is generally assumed that there are only two ways to maintain the ends of chromosomes in yeast and mammalian nuclei: telomerase and recombination . Without telomerase and recombination, cells enter senescence, a state of permanent growth arrest . We found that the decisive role in preventing senescent budding yeast cells from dividing is played by the Exo1 nuclease . In the absence of Exo1, telomerase- and recombination-defective yeast can resume cell cycle progression, despite degradation of telomeric regions from many chromosomes . As degradation progresses toward internal chromosomal regions, a progressive decrease in viability would be expected, caused by loss of essential genes . However, this was not the case . We demonstrate that extensive degradation and loss of essential genes can be efficiently prevented through a little-studied mechanism of DNA double-strand-break repair, in which short DNA palindromes induce formation of large DNA palindromes . For the first time, we show that large palindromes form as a natural consequence of postsenescence growth and that they become essential for immortalization in the absence of telomerase activity.

Proc Natl Acad Sci U S A, 2004 Oct 26, 101(43), 15392 - 7 Epub 2004 Oct 15.
Gene targeting in yeast is initiated by two independent strand invasions; Langston LD et al.; To study the mechanism of gene targeting, we examined heteroduplex DNA (hDNA) formation during targeting of two separate chromosomal locations in Saccharomyces cerevisiae . We examined both replacement of the entire gene with a heterologous selectable marker and correction of a single base pair insertion mutation by gene targeting, and in all cases our results were consistent with separate strand invasion/resolution at the two ends of the targeting fragment as the dominant mechanism in wild-type cells . A small subset of transformants was consistent with assimilation of a single strand of targeting DNA encompassing both flanking homology regions and the marker into hDNA . hDNA formation during correction of a point mutation by targeted integration was conspicuously altered in a mismatch repair-deficient background and was consistent with single-strand invasion/assimilation without mismatch correction, confirming that gene targeting by this pathway is actively impeded in wild-type yeast . Finally, inversion of one targeted locus and mutation of an active origin of DNA replication at the other locus affected hDNA formation significantly, suggesting that formation of productive interactions between the targeting DNA and the targeted site in the chromosome is sensitive to local DNA dynamics.

FEMS Yeast Res, 2004 Nov, 5(2), 157 - 67
The role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway; Heeren G et al.; We show that the dominant activated allele of the yeast RAS gene, RAS2(ala18,val19), led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase . The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cell-specific lifespan . Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan . The corresponding RAS2(+) rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR . Adding 1 mM GSH completely restored the lifespan of the RAS2(+) rho-zero strain to that of the wild-type cells . The double mutant RAS2(ala18,val19) rho-zero cells showed the same lifespan as the RAS2(ala18,val19) cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism . In the RAS2(ala18,val19) mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion . The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation . Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.

FEMS Yeast Res, 2004 Nov, 5(2), 149 - 56
Production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of Bcl-xL; Trancikova A et al.; The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells . The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain . Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase . The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria . Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation . If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event . The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L).

FEMS Yeast Res, 2004 Nov, 5(2), 141 - 7
Involvement of the yeast metacaspase Yca1 in ubp10Delta-programmed cell death; Bettiga M et al.; UBP10 encodes a deubiquitinating enzyme of Saccharomyces cerevisiae . Its inactivation results in a complex phenotype characterized by a subpopulation of cells that exhibits the typical cellular markers of apoptosis . Here, we show that additional deletion of YCA1, coding for the yeast metacaspase, suppressed the ubp10 disruptant phenotype . Moreover, YCA1 overexpression, without any external stimulus, had a detrimental effect on growth and viability of ubp10 cells accompanied by an increase of apoptotic cells . This response was completely abrogated by ascorbic acid addition . We also observed that cells lacking UBP10 had an endogenous caspase activity, revealed by incubation in vivo with FITC-labeled VAD-fmk . All these results argue in favour of an involvement of the yeast metacaspase in the active cell death triggered by loss of UBP10 function.

FEMS Yeast Res, 2004 Nov, 5(2), 133 - 40
Uth1p: a yeast mitochondrial protein at the crossroads of stress, degradation and cell death; Camougrand N et al.; UTH1 is a yeast aging gene that has been identified on the basis of stress resistance and longer life span of mutants . It was also shown to participate in mitochondrial biogenesis . The absence of Uth1p was found to trigger resistance to autophagy induced by rapamycin . Uth1p is therefore the first mitochondrial protein proven to be required for the autophagic degradation of mitochondria . Since this protein is also involved in yeast cell death induced by heterologous expression of the pro-apoptotic protein Bax, the results are discussed in the light of evidence suggesting a co-regulation of apoptosis and autophagy in mammalian cells.

FEMS Yeast Res, 2004 Nov, 5(2), 119 - 25
Yeast replicative life span--the mitochondrial connection; Jazwinski SM; Mitochondria have been associated with aging in many experimental systems through the damaging action of reactive oxygen species . There is more, however, to the connection between mitochondria and Saccharomyces cerevisiae longevity and aging . Induction of the retrograde response, a pathway signaling mitochondrial dysfunction, results in the extension of life span and postponement of the manifestations of aging, changing the metabolic and stress resistance status of the cell . A paradox associated with the retrograde response is the simultaneous triggering of extrachromosomal ribosomal DNA circle (ERC) production, because of the deleterious effect these circles have on yeast longevity . The retrograde response gene RTG2 appears to play a pivotal role in ERC production, linking metabolism and genome stability . In addition to mother cell aging, mitochondria are important in establishment of age asymmetry between mother and daughter cells . The results more generally point to the existence of a mechanism to "filter" damaged components from daughter cells, a form of checkpoint control . Mitochondrial integrity is affected by the PHB1 and PHB2 genes, which encode inner mitochondrial membrane chaperones called prohibitins . The Phb1/2 proteins protect the cell from imbalances in the production of mitochondrial proteins . Such imbalances appear to cause a stochastic stratification of the yeast population with the appearance of short-lived cells . Ras2p impacts this process . Maintenance of mitochondrial membrane potential and the provision of Krebs cycle intermediates for biosyntheses appear to be crucial elements in yeast longevity . In sum, it is clear that mitochondria lie at the nexus of yeast longevity and aging.

FEMS Yeast Res, 2004 Nov, 5(2), 111 - 7
Programmed cell death in fission yeast; Rodriguez-Menocal L et al.; Recently a metacaspase, encoded by YCA1, has been implicated in a primitive form of apoptosis or programmed cell death in yeast . Previously it had been shown that over-expression of mammalian pro-apoptotic proteins can induce cell death in yeast, but the mechanism of how cell death occurred was not clearly established . More recently, it has been shown that DNA or oxidative damage, or other cell cycle blocks, can result in cell death that mimics apoptosis in higher cells . Also, in fission yeast deletion of genes required for triacylglycerol synthesis leads to cell death and expression of apoptotic markers . A metacaspase sharing greater than 40% identity to budding yeast Yca1 has been identified in fission yeast, however, its role in programmed cell death is not yet known . Analysis of the genetic pathways that influence cell death in yeast may provide insights into the mechanisms of apoptosis in all eukaryotic organisms.

Arch Biochem Biophys, 2004 Nov 15, 431(2), 207 - 14
Expression of a familial amyotrophic lateral sclerosis-associated mutant human superoxide dismutase in yeast leads to decreased mitochondrial electron transport; Gunther MR et al.; Strains of Saccharomyces cerevisiae that express either the wild type or the amyotrophic lateral sclerosis-associated mutant human copper-zinc superoxide dismutase (SOD1) proteins A4V and G93A, respectively, in a yeast SOD1-deficient parent strain were used to investigate the hypothesis that expression of a mutant SOD1 protein causes deficient mitochondrial electron transport as a possible mechanism for disease induction . Mitochondria isolated from the wild type SOD1-expressing yeast were identical to mitochondria from the parent strain in heme content and activities of complexes II, III, and IV . Mitochondria isolated from the A4V-expressing yeast had decreased rates of electron transport in complexes II+III, III, and IV and corresponding decreases in hemes b, c-c1, and a-a3 content compared to mitochondria from wild type human SOD1-expressing yeast . Mitochondria isolated from G93A-expressing yeast had decreased rates of electron transport in complex IV and probably in complex II with a corresponding decrease in heme a-a3 content . These results suggest that mutant SOD1-expression causes defective electron transport complex assembly and that the yeast system will provide an excellent model for the study of the mechanism of mutant SOD1-induced mitochondrial electron transport defects.

J Trace Elem Med Biol, 2004, 18(1), 47 - 51
Chromium-yeast supplementation of chicken broilers in an industrial farming system; Debski B et al.; The experiment was performed on ISA broilers at big farm conditions . One day old chickens were randomly assigned to two poultry-houses with the capacity of 10600 birds each . Chromium was provided in the form of chromium enriched yeast (1.0 g Cr/kg yeast) given in doses of 0.2 mg Cr/kg diet . No significant differences in growth rate and only a slight decrease of feed conversion ratio was found in broilers receiving chromium-yeast supplement . However, these birds had a lower mortality rate comparied with the rate observed in the controls . Moreover, dietary chromium led also to an improvement of carcass composition . Organic chromium supplementation increased the weight of pectoral muscles and meat of these broilers contained less amounts of fat and cholesterol . These effects were accompanied by an insignificant increase of chromium concentration in the pectoral muscles of birds fed chromium supplemented diets, from 40 to 52 microg/kg wet tissue.

Mol Cell Biol, 2004 Nov, 24(21), 9646 - 57
Nuclear pre-mRNA decapping and 5' degradation in yeast require the Lsm2-8p complex; Kufel J et al.; Previous analyses have identified related cytoplasmic Lsm1-7p and nuclear Lsm2-8p complexes . Here we report that mature heat shock and MET mRNAs that are trapped in the nucleus due to a block in mRNA export were strongly stabilized in strains lacking Lsm6p or the nucleus-specific Lsm8p protein but not by the absence of the cytoplasmic Lsm1p . These nucleus-restricted mRNAs remain polyadenylated until their degradation, indicating that nuclear mRNA degradation does not involve the incremental deadenylation that is a key feature of cytoplasmic turnover . Lsm8p can be UV cross-linked to nuclear poly(A)(+) RNA, indicating that an Lsm2-8p complex interacts directly with nucleus-restricted mRNA . Analysis of pre-mRNAs that contain intronic snoRNAs indicates that their 5' degradation is specifically inhibited in strains lacking any of the Lsm2-8p proteins but Lsm1p . Nucleus-restricted mRNAs and pre-mRNA degradation intermediates that accumulate in lsm mutants remain 5' capped . We conclude that the Lsm2-8p complex normally targets nuclear RNA substrates for decapping.

J Biol Chem, 2004 Dec 17, 279(51), 53707 - 16 Epub 2004 Oct 12.
The topology of the Lcb1p subunit of yeast serine palmitoyltransferase; Han G et al.; The structural organization and topology of the Lcb1p subunit of yeast and mammalian serine palmitoyltransferases (SPT) were investigated . In the yeast protein, three membrane-spanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions . The first domain of the yeast protein, located between residues 50 and 84, was not required for the stability, membrane association, interaction with Lcb2p, or enzymatic activity . Deletion of the comparable domain of the mammalian protein SPTLC1 also had little effect on its function, demonstrating that this region is not required for membrane localization or heterodimerization with SPTLC2 . The second and third membrane-spanning domains of yeast Lcb1p, located between residues 342 and 371 and residues 425 and 457, respectively, create a luminal loop of approximately 60 residues . In contrast to the first membrane-spanning domain, the second and third membrane-spanning domains were both required for Lcb1p stability . In addition, mutations in the luminal loop destabilized the SPT heterodimer indicating that this region of the protein is important for SPT structure and function . Mutations in the extreme carboxyl-terminal region of Lcb1p also disrupted heterodimer formation . Taken together, these data suggest that in contrast to other members of the alpha-oxoamine synthases that are soluble homodimers, the Lcb1p and Lcb2p subunits of the SPT heterodimer may interact in the cytosol, as well as within the membrane and/or the lumen of the endoplasmic reticulum.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Oct, 24(10), 1150 - 2
{A new method for screening mutant yeast strains with green fluorescent protein.}; Hu LM et al.; OBJECTIVE: To establish a fast and simple method for screening mutant yeast strains . MATERIALS AND METHODS: Homologous recombination technique was used to detect mutant yeast strains in yeast genomic library, with the green fluorescent protein gene as the reporter gene in the transposon . RESULTS: The strains that emitted green fluorescence were isolated, indicating that the gfp gene was inserted into the yeast genome by homologous recombination . CONCLUSION: This study established a useful method for functional genome study by homologous recombination technique, and provide an alternative for gene therapeutic drug development.

Biochem Biophys Res Commun, 2004 Nov 19, 324(3), 1101 - 7
The herbicide 2,4-dichlorophenoxyacetic acid induces the generation of free-radicals and associated oxidative stress responses in yeast; Teixeira MC et al.; The pro-oxidant action of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is demonstrated in this study using Saccharomyces cerevisiae as a eukaryotic experimental model . Evidence is presented for the generation of hydroxyl-radicals, in yeast cells suddenly exposed to 2,4-D, detected by in vivo electron paramagnetic resonance (EPR) spectroscopy using 5,5'-dimethyl-1-pyrroline N-oxide and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide as spin-traps . The intensity of the EPR spectra was dependent on the concentration of herbicide tested and was consistently higher in a mutant (Deltasod1) devoid of the cytosolic CuZn-superoxide dismutase . A time-course-dependent variation of the level of free-radical adducts was registered upon sudden exposure of an yeast cell population to concentrations of 2,4-D that lead to an initial period of viability loss, before resumption of inhibited growth by the viable adapted population . The variation pattern of the level of hydroxyl-radical adducts correlated with the one determined for the activity of Sod1p, cytosolic catalase Ctt1p, and the dithiol glutaredoxins Grx1p and Grx2p.

Mol Biol Cell, 2004 Dec, 15(12), 5492 - 502 Epub 2004 Dec.
Cold adaptation in budding yeast; Schade B et al.; We have determined the transcriptional response of the budding yeast Saccharomyces cerevisiae to cold . Yeast cells were exposed to 10 degrees C for different lengths of time, and DNA microarrays were used to characterize the changes in transcript abundance . Two distinct groups of transcriptionally modulated genes were identified and defined as the early cold response and the late cold response . A detailed comparison of the cold response with various environmental stress responses revealed a substantial overlap between environmental stress response genes and late cold response genes . In addition, the accumulation of the carbohydrate reserves trehalose and glycogen is induced during late cold response . These observations suggest that the environmental stress response (ESR) occurs during the late cold response . The transcriptional activators Msn2p and Msn4p are involved in the induction of genes common to many stress responses, and we show that they mediate the stress response pattern observed during the late cold response . In contrast, classical markers of the ESR were absent during the early cold response, and the transcriptional response of the early cold response genes was Msn2p/Msn4p independent . This implies that the cold-specific early response is mediated by a different and as yet uncharacterized regulatory mechanism.

Mol Biol Cell, 2005 Jan, 16(1), 316 - 27 Epub 2004 Oct 13.
BAF53/Arp4 Homolog Alp5 in Fission Yeast Is Required for Histone H4 Acetylation, Kinetochore-Spindle Attachment, and Gene Silencing at Centromere; Minoda A et al.; Nuclear actin-related proteins play vital roles in transcriptional regulation; however, their biological roles remain elusive . Here, we characterize Alp5, fission yeast homolog of Arp4/BAF53 . The temperature-sensitive mutant alp5-1134 contains a single amino acid substitution in the conserved C-terminal domain (S402N) and displays mitotic phenotypes, including chromosome condensation and missegregation . Alp5 forms a complex with Mst1-HAT (histone acetyltransferase) . Consistently, inhibition of histone deacetylases (HDACs), by either addition of a specific inhibitor or a mutation in HDAC-encoding clr6(+) gene, rescues alp5-1134 . Immunoblotting with specific antibodies against acetylated histones shows that Alp5 is required for histone H4 acetylation at lysines 5, 8, and 12, but not histone H3 lysines 9 or 14, and furthermore Clr6 plays an opposing role . Mitotic arrest is ascribable to activation of the Mad2/Bub1 spindle checkpoint, in which both proteins localize to the mitotic kinetochores in alp5-1134 . Intriguingly, alp5-1134 displays transcriptional desilencing at the core centromere without altering the overall chromatin structure, which also is suppressed by a simultaneous mutation in clr6(+) . This result shows that Alp5 is essential for histone H4 acetylation, and its crucial role lies in the establishment of bipolar attachment of the kinetochore to the spindle and transcriptional silencing at the centromere.

J Cell Biol, 2004 Oct 11, 167(1), 87 - 98
Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway; Frederick RL et al.; Cell signaling events elicit changes in mitochondrial shape and activity . However, few mitochondrial proteins that interact with signaling pathways have been identified . Candidates include the conserved mitochondrial Rho (Miro) family of proteins, which contain two GTPase domains flanking a pair of calcium-binding EF-hand motifs . We show that Gem1p (yeast Miro; encoded by YAL048C) is a tail-anchored outer mitochondrial membrane protein . Cells lacking Gem1p contain collapsed, globular, or grape-like mitochondria . We demonstrate that Gem1p is not an essential component of characterized pathways that regulate mitochondrial dynamics . Genetic studies indicate both GTPase domains and EF-hand motifs, which are exposed to the cytoplasm, are required for Gem1p function . Although overexpression of a mutant human Miro protein caused increased apoptotic activity in cultured cells (Fransson et al., 2003 . J . Biol . Chem . 278:6495-6502), Gem1p is not required for pheromone-induced yeast cell death . Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics.

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